Human Anti-OPGL Neutralizing Antibodies as Selective OPGL Pathway Inhibitors

ABSTRACT

Monoclonal antibodies and hybridomas producing them that interact with osteoprotegerin ligand (OPGL) are provided. Methods of treating osteopenic disorders by administering a pharmaceutically effective amount of antibodies to OPGL are also provided. Methods of detecting the amount of OPGL in a sample using antibodies to OPGL are further provided.

This application is a divisional application of U.S. non-provisionalapplication Ser. No. 10/408,901, filed Apr. 7, 2003, now U.S. Pat. No.7,718,776, which is related to and claims priority to U.S. provisionalapplication Ser. No. 60/370,407, filed Apr. 5, 2002. The disclosure ofeach of these documents is incorporated by reference herein.

FIELD OF THE INVENTION

The invention relates to antibodies that bind osteoprotegerin ligand(OPGL). Compositions and methods for the treatment of bone diseases,such as osteoporosis, bone loss from arthritis, Paget's disease, andosteopenia, are also provided.

BACKGROUND OF THE INVENTION

Living bone tissue exhibits a dynamic equilibrium between formation ofbone, known as deposition, and breakdown of bone, known as resorption.These processes can be mediated by at least two cell types: osteoblasts,which secrete molecules that comprise the organic matrix of bone(deposition); and osteoclasts, which promote dissolution of the bonematrix and solubilization of bone salts (resorption). In certainindividuals, such as post-menopausal women, the rate of resorption canexceed the rate of deposition, which may result in reduced bone mass andstrength, increased risk of fractures, and slow or incomplete repair ofbroken bones.

Osteoprotegerin ligand (OPGL) is a member of the TNF family of cytokinesand promotes formation of osteoclasts through binding to the receptoractivator of NF-κB (RANK, also called osteoclast differentiation andactivation receptor, or ODAR). Osteoprotegerin (OPG), on the other hand,inhibits the formation of osteoclasts by sequestering OPGL andpreventing OPGL association with ODAR. Thus, the amount of OPGLassociated with ODAR correlates with the equilibrium between bonedeposition and resorption. Individuals who suffer from osteopenicdiseases, such as osteoporosis, show a greater rate of bone resorptionthan deposition, which may result from increased levels or activity ofOPGL. Thus, it would be useful to have molecules that can regulate theactivity of OPGL in osteoclastogenesis. It would also be useful to beable to detect the amount of OPGL in a biological sample, such as ablood sample, to diagnose an osteopenic disorder relating to increasedlevels of OPGL.

SUMMARY OF THE INVENTION

The invention provides monoclonal antibodies that bind toosteoprotegerin ligand (OPGL). Preferably, the antibodies inhibitbinding of OPGL to an osteoclast differentiation and activation receptor(ODAR). Also provided by this invention are hybridoma cell lines thatproduce, and most preferably, secrete into cell culture media themonoclonal antibodies of the invention. The antibodies of the inventionare useful for treating various disorders associated with low bonedensity.

In certain aspects, the invention provides antibodies, preferablymonoclonal antibodies, most preferably human antibodies, comprising aheavy chain and a light chain, wherein the heavy chain comprises anIgG₁, IgG₂, or an IgG₄ heavy chain constant region. Preferably, anantibody of the invention comprises an amino acid sequence of the IgG₁heavy chain constant region as set forth in SEQ ID NO: 2 or anantigen-binding or an immunologically functional immunoglobulin fragmentthereof.

The invention also provides antibodies, preferably monoclonalantibodies, most preferably human antibodies, comprising a heavy chainand a light chain, wherein the light chain comprises an amino acidsequence as set forth in SEQ ID NO: 4 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof.

The invention relates specifically to human antibodies, most preferablymonoclonal antibodies that specifically bind the D-E loop region ofOPGL. The invention also relates to human antibodies, preferablymonoclonal antibodies, that bind to a region of osteoprotegerin ligand(OPGL) that is outside the D-E loop region. In addition, the inventionrelates to human antibodies, preferably monoclonal antibodies, that bindto both a region of OPGL that is outside the D-E loop region and all ora portion of the D-E loop region. In one aspect, antibodies of theinvention bind to a first region of OPGL that is outside the D-E loopregion and then, while remaining bound to the first region, bind to asecond region that is all or a portion of the D-E loop region. Suchbinding is referred to herein as consecutive. In another aspect,antibodies of the invention can bind to a first region of OPGL that isoutside the D-E loop region and a second region that is all or a portionof the D-E loop region at the same time. Such binding is referred toherein as simultaneous.

In certain aspects, antibodies of the invention comprise a heavy chainand a light chain, wherein the variable region of the heavy chaincomprises an amino acid sequence as set forth in any of SEQ ID NO: 6,SEQ ID NO: 14, SEQ ID NO: 22, or SEQ ID NO: 26, or an antigen-binding oran immunologically functional immunoglobulin fragment thereof. In otheraspects, the light chain variable region comprises an amino acidsequence as set forth in any of SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO:24, or SEQ ID NO: 28, or an antigen-binding or an immunologicallyfunctional immunoglobulin fragment thereof. In additional aspects, theheavy chain comprises an amino acid sequence as set forth in any of SEQID NO: 30, SEQ ID NO: 38, SEQ ID NO: 46, or SEQ ID NO: 50, or anantigen-binding or an immunologically functional immunoglobulin fragmentthereof. In still further aspects, the light chain comprises an aminoacid sequence as set forth in any of SEQ ID NO: 32, SEQ ID NO: 40, SEQID NO: 48, or SEQ ID NO: 52, or an antigen-binding or an immunologicallyfunctional immunoglobulin fragment thereof.

The invention also provides antibodies that bind specifically to OPGL,wherein the heavy chain comprises a heavy chain variable region as setforth in SEQ ID NO: 6, or an antigen-binding or an immunologicallyfunctional immunoglobulin fragment thereof, and the light chaincomprises a light chain variable region comprising an amino acidsequence as set forth in SEQ ID NO: 8, or an antigen-binding or animmunologically functional immunoglobulin fragment thereof.

In certain aspects, the invention provides antibodies, comprising aheavy chain and a light chain, (a) wherein the heavy chain comprises afirst variable region, and wherein the first variable region comprises asequence that has at least 90% identity to the amino acid sequence setforth in SEQ ID NO: 6, and (b) wherein the light chain comprises asecond variable region, and wherein the second variable region comprisesa sequence that has at least 90% identity to the amino acid sequence setforth in SEQ ID NO: 8, and (c) wherein the antibody interacts with OPGL.

In other aspects, the first variable region comprises a sequence thathas at least 95% identity to the amino acid sequence set forth in SEQ IDNO: 6, and the second variable region comprises a sequence that has atleast 95% identity to the amino acid sequence set forth in SEQ ID NO: 8.

In still other aspects, the first variable region comprises a sequencethat has at least 99% identity to the amino acid sequence set forth inSEQ ID NO: 6, and the second variable region comprises a sequence thathas at least 99% identity to the amino acid sequence set forth in SEQ IDNO: 8.

The invention further provides antibodies that bind specifically toOPGL, wherein the heavy chain comprises a heavy chain variable region asset forth in SEQ ID NO: 14, or an antigen-binding or an immunologicallyfunctional immunoglobulin fragment thereof, and the light chaincomprises a light chain variable region comprising an amino acidsequence as set forth in SEQ ID NO: 16, or an antigen-binding or animmunologically functional immunoglobulin fragment thereof.

In certain aspects, the invention provides antibodies, comprising aheavy chain and a light chain, (a) wherein the heavy chain comprises afirst variable region, and wherein the first variable region comprises asequence that has at least 90% identity to the amino acid sequence setforth in SEQ ID NO: 14, and (b) wherein the light chain comprises asecond variable region, and wherein the second variable region comprisesa sequence that has at least 90% identity to the amino acid sequence setforth in SEQ ID NO: 16, and (c) wherein the antibody interacts withOPGL.

In other aspects, the first variable region comprises a sequence thathas at least 95% identity to the amino acid sequence set forth in SEQ IDNO: 14, and the second variable region comprises a sequence that has atleast 95% identity to the amino acid sequence set forth in SEQ ID NO:16.

In further aspects, the first variable region comprises a sequence thathas at least 99% identity to the amino acid sequence set forth in SEQ IDNO: 14, and the second variable region comprises a sequence that has atleast 99% identity to the amino acid sequence set forth in SEQ ID NO:16.

The invention provides antibodies that bind specifically to OPGL,wherein the heavy chain comprises a heavy chain variable region as setforth in SEQ ID NO: 22, or an antigen-binding or an immunologicallyfunctional immunoglobulin fragment thereof, and the light chaincomprises a light chain variable region comprising an amino acidsequence as set forth in SEQ ID NO: 24, or an antigen-binding or animmunologically functional immunoglobulin fragment thereof.

In certain aspects, the invention provides antibodies, comprising aheavy chain and a light chain, (a) wherein the heavy chain comprises afirst variable region, and wherein the first variable region comprises asequence that has at least 90% identity to the amino acid sequence setforth in SEQ ID NO: 22, and (b) wherein the light chain comprises asecond variable region, and wherein the second variable region comprisesa sequence that has at least 90% identity to the amino acid sequence setforth in SEQ ID NO: 24, and (c) wherein the antibody interacts withOPGL.

In particular aspects, the first variable region comprises a sequencethat has at least 95% identity to the amino acid sequence set forth inSEQ ID NO: 22, and the second variable region comprises a sequence thathas at least 95% identity to the amino acid sequence set forth in SEQ IDNO: 24.

In further aspects, the first variable region comprises a sequence thathas at least 99% identity to the amino acid sequence set forth in SEQ IDNO: 22, and the second variable region comprises a sequence that has atleast 99% identity to the amino acid sequence set forth in SEQ ID NO:24.

In addition, the invention provides antibodies that bind specifically tothe D-E loop region of OPGL, wherein the heavy chain comprises a heavychain variable region as set forth in SEQ ID NO: 26, or anantigen-binding or an immunologically functional immunoglobulin fragmentthereof, and the light chain comprises a light chain variable regioncomprising an amino acid sequence as set forth in SEQ ID NO: 28, or anantigen-binding or an immunologically functional immunoglobulin fragmentthereof.

In certain aspects, the invention provides antibodies, comprising aheavy chain and a light chain, (a) wherein the heavy chain comprises afirst variable region, and wherein the first variable region comprises asequence that has at least 90% identity to the amino acid sequence setforth in SEQ ID NO: 26, and (b) wherein the light chain comprises asecond variable region, and wherein the second variable region comprisesa sequence that has at least 90% identity to the amino acid sequence setforth in SEQ ID NO: 28, and (c) wherein the antibody interacts withOPGL.

In other aspects, the first variable region comprises a sequence thathas at least 95% identity to the amino acid sequence set forth in SEQ IDNO: 26, and the second variable region comprises a sequence that has atleast 95% identity to the amino acid sequence set forth in SEQ ID NO:28.

In additional aspects, the first variable region comprises a sequencethat has at least 99% identity to the amino acid sequence set forth inSEQ ID NO: 26, and the second variable region comprises a sequence thathas at least 99% identity to the amino acid sequence set forth in SEQ IDNO: 28.

The invention also provides antibodies that bind specifically to OPGL,wherein the heavy chain comprises an amino acid sequence as set forth inSEQ ID NO: 30, or an antigen-binding or an immunologically functionalimmunoglobulin fragment thereof, and the light chain comprises an aminoacid sequence as set forth in SEQ ID NO: 32, or an antigen-binding or animmunologically functional immunoglobulin fragment thereof.

The invention also provides antibodies that bind specifically to OPGL,wherein the heavy chain comprises an amino acid sequence as set forth inSEQ ID NO: 38, or an antigen-binding or an immunologically functionalimmunoglobulin fragment thereof, and the light chain comprises a lightchain variable region comprising an amino acid sequence as set forth inSEQ ID NO: 40, or an antigen-binding or an immunologically functionalimmunoglobulin fragment thereof.

The invention provides antibodies that bind specifically to OPGL,wherein the heavy chain comprises an amino acid sequence as set forth inSEQ ID NO: 46, or an antigen-binding or an immunologically functionalimmunoglobulin fragment thereof, and the light chain comprises a lightchain variable region comprising an amino acid sequence as set forth inSEQ ID NO: 48, or an antigen-binding or an immunologically functionalimmunoglobulin fragment thereof.

The invention provides antibodies that bind specifically to OPGL,wherein the heavy chain comprises an amino acid sequence as set forth inSEQ ID NO: 50, or an antigen-binding or an immunologically functionalimmunoglobulin fragment thereof, and the light chain comprises a lightchain variable region comprising an amino acid sequence as set forth inSEQ ID NO: 52, or an antigen-binding or an immunologically functionalimmunoglobulin fragment thereof.

In certain aspects, the invention provides antibodies that specificallybind OPGL and comprises a heavy chain and a light chain, wherein theheavy chain variable region comprises an amino acid sequence as setforth in SEQ ID NO: 10 or SEQ ID NO: 18, or an antigen-binding or animmunologically functional immunoglobulin fragment thereof. In otheraspects, the light chain variable region comprises an amino acidsequence as set forth in SEQ ID NO: 12 or SEQ ID NO: 20, or anantigen-binding or an immunologically functional immunoglobulin fragmentthereof.

The invention also provides antibodies that specifically bind OPGL,wherein the heavy chain variable region comprises an amino acid sequenceas set forth in SEQ ID NO: 34 or SEQ ID NO: 42, or an antigen-binding oran immunologically functional immunoglobulin fragment thereof. In otheraspects, the light chain variable region comprising an amino acidsequence as set forth in SEQ ID NO: 36 or SEQ ID NO: 44, or anantigen-binding or an immunologically functional immunoglobulin fragmentthereof.

The invention further provides antibodies that specifically bind OPGL,wherein the heavy chain comprises a heavy chain variable region as setforth in SEQ ID NO: 10, or an antigen-binding or an immunologicallyfunctional immunoglobulin fragment thereof, and the light chaincomprises a light chain variable region comprising an amino acidsequence as set forth in SEQ ID NO: 12, or an antigen-binding or animmunologically functional immunoglobulin fragment thereof.

In certain aspects, the invention provides antibodies, comprising aheavy chain and a light chain, (a) wherein the heavy chain comprises afirst variable region, and wherein the first variable region comprises asequence that has at least 90% identity to the amino acid sequence setforth in SEQ ID NO: 10, and (b) wherein the light chain comprises asecond variable region, and wherein the second variable region comprisesa sequence that has at least 90% identity to the amino acid sequence setforth in SEQ ID NO: 12, and (c) wherein the antibody interacts withOPGL.

In further aspects, the first variable region comprises a sequence thathas at least 95% identity to the amino acid sequence set forth in SEQ IDNO: 10, and the second variable region comprises a sequence that has atleast 95% identity to the amino acid sequence set forth in SEQ ID NO:12.

In other aspects, the first variable region comprises a sequence thathas at least 99% identity to the amino acid sequence set forth in SEQ IDNO: 10, and the second variable region comprises a sequence that has atleast 99% identity to the amino acid sequence set forth in SEQ ID NO:12.

The invention also provides antibodies that specifically bind, whereinthe heavy chain comprises a heavy chain variable region as set forth inSEQ ID NO: 18, or an antigen-binding or an immunologically functionalimmunoglobulin fragment thereof, and the light chain comprises a lightchain variable region comprising an amino acid sequence as set forth inSEQ ID NO: 20, or an antigen-binding or an immunologically functionalimmunoglobulin fragment thereof.

In certain aspects, the invention provides antibodies, comprising aheavy chain and a light chain, (a) wherein the heavy chain comprises afirst variable region, and wherein the first variable region comprises asequence that has at least 90% identity to the amino acid sequence setforth in SEQ ID NO: 18, and (b) wherein the light chain comprises asecond variable region, and wherein the second variable region comprisesa sequence that has at least 90% identity to the amino acid sequence setforth in SEQ ID NO: 20, and (c) wherein the antibody interacts withOPGL.

In other aspects, the first variable region comprises a sequence thathas at least 95% identity to the amino acid sequence set forth in SEQ IDNO: 18, and the second variable region comprises a sequence that has atleast 95% identity to the amino acid sequence set forth in SEQ ID NO:20.

In still other aspects, the first variable region comprises a sequencethat has at least 99% identity to the amino acid sequence set forth inSEQ ID NO: 18, and the second variable region comprises a sequence thathas at least 99% identity to the amino acid sequence set forth in SEQ IDNO: 20.

The invention also provides antibodies that specifically bind OPGL,wherein the heavy chain comprises an amino acid sequence as set forth inSEQ ID NO: 34, or an antigen-binding or an immunologically functionalimmunoglobulin fragment thereof, and the light chain comprises a lightchain variable region comprising an amino acid sequence as set forth inSEQ ID NO: 36, or an antigen-binding or an immunologically functionalimmunoglobulin fragment thereof.

The invention provides antibodies that specifically bind OPGL, whereinthe heavy chain comprises an amino acid sequence as set forth in SEQ IDNO: 42, or an antigen-binding or an immunologically functionalimmunoglobulin fragment thereof, and the light chain comprises a lightchain variable region comprising an amino acid sequence as set forth inSEQ ID NO: 44, or an antigen-binding or an immunologically functionalimmunoglobulin fragment thereof.

The invention also provides single chain antibodies, single chain Fvantibodies, Fab antibodies, Fab′ antibodies, and (Fab′)₂.

In particular aspects, the invention provides a heavy chain comprising avariable region and a constant region, wherein the variable regioncomprises an amino acid sequence as set forth in any of SEQ ID NO: 6,SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, or SEQ IDNO: 26, or an antigen-binding or an immunologically functionalimmunoglobulin fragment thereof.

In addition, the invention also provides a heavy chain comprising anamino acid sequence as set forth in any of SEQ ID NO: 30, SEQ ID NO: 34,SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 46, or SEQ ID NO: 50, or anantigen-binding or an immunologically functional immunoglobulin fragmentthereof.

In certain aspects, the invention provides a light chain comprising avariable region and a constant region, wherein the variable regioncomprises an amino acid sequence as set forth in any of SEQ ID NO: 8,SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, or SEQ IDNO: 28, or an antigen-binding or an immunologically functionalimmunoglobulin fragment thereof.

In other aspects, the invention provides a light chain comprising anamino acid sequence as set forth in any of SEQ ID NO: 32, SEQ ID NO: 36,SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 48, or SEQ ID NO: 52, or anantigen-binding or an immunologically functional immunoglobulin fragmentthereof.

The invention also relates to isolated human antibodies thatspecifically bind OPGL, wherein the antibody comprises: (a) human heavychain framework regions, a human heavy chain CDR1 region, a human heavychain CDR2 region, and a human heavy chain CDR3 region; and (b) humanlight chain framework regions, a human light chain CDR1 region, a humanlight chain CDR2 region, and a human light chain CDR3 region. In certainaspects, the human heavy chain CDR1 region can be the heavy chain CDR1region of 16E1, 2D8, 22B3, or 9H7 as shown in FIG. 15 and the humanlight chain CDR1 region can be the light chain CDR1 region of 16E1, 2D8,22B3, or 9H7 as shown in FIG. 16. In other aspects, the human heavychain CDR2 region can be the heavy chain CDR2 region of 16E1, 2D8, 22B3,or 9H7 as shown in FIG. 15 and the human light chain CDR2 region can bethe light chain CDR2 region of 16E1, 2D8, 22B3, or 9H7 as shown in FIG.16. In still other aspects, the human heavy chain CDR3 region is theheavy chain CDR3 region of 16E1, 2D8, 22B3, or 9H7 as shown in FIG. 15,and the human light chain CDR3 region is the light chain CDR3 region of16E1, 2D8, 22B3, or 9H7 as shown in FIG. 16.

The invention also relates to isolated human antibodies thatspecifically bind OPGL, wherein the antibody comprises: (a) human heavychain framework regions, a human heavy chain CDR1 region, a human heavychain CDR2 region, and a human heavy chain CDR3 region; and (b) humanlight chain framework regions, a human light chain CDR1 region, a humanlight chain CDR2 region, and a human light chain CDR3 region. In certainaspects, the human heavy chain CDR1 region can be the heavy chain CDR1region of 2E11 or 18B2 as shown in FIG. 15 and the human light chainCDR1 region can be the light chain CDR1 region of 2E11 or 18B2 as shownin FIG. 16. In other aspects, the human heavy chain CDR2 region can bethe heavy chain CDR2 region of 2E11 or 18B2 as shown in FIG. 15 and thehuman light chain CDR2 region can be the light chain CDR2 region of 2E11or 18B2 as shown in FIG. 16. In still other aspects, the human heavychain CDR3 region is the heavy chain CDR3 region of 2E11 or 18B2 asshown in FIG. 15, and the human light chain CDR3 region is the lightchain CDR3 region of 2E11 or 18B2 as shown in FIG. 16.

In addition, the invention provides methods for treating an osteopenicdisorder, comprising the step of administering a pharmaceuticallyeffective amount of a monoclonal antibody of the invention orantigen-binding fragment thereof to an individual in need thereof.

The invention further relates to fusion proteins and other moleculescapable of binding to a region of osteoprotegerin ligand (OPGL) that isoutside the D-E loop region, or both a region of OPGL that is outsidethe D-E loop region and all or a portion of the D-E loop region, whereinbinding is consecutive or simultaneous (together with the aforementionedantibodies, collectively referred to herein as “specific bindingpartners”), such as may be prepared using methods as described, forexample, in WO 00/24782, which is incorporated by reference. Suchmolecules can be expressed, for example, in mammalian cells (e.g.Chinese Hamster Ovary cells) or bacterial cells (e.g. E. coli cells).

The invention also provides methods for detecting the level of OPGL in abiological sample, comprising the step of contacting the sample with amonoclonal antibody of the invention or antigen-binding fragmentthereof. The anti-OPGL antibodies of the invention may be employed inany known assay method, such as competitive binding assays, direct andindirect sandwich assays, immunoprecipitation assays and enzyme-linkedimmunosorbent assays (ELISA) (See, Sola, 1987, Monoclonal Antibodies: AManual of Techniques, pp. 147-158, CRC Press, Inc.) for the detectionand quantitation of OPGL. The antibodies can bind OPGL with an affinitythat is appropriate for the assay method being employed.

Specific preferred embodiments of the present invention will becomeevident from the following more detailed description of certainpreferred embodiments and the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B depict a cDNA sequence (FIG. 1A) encoding the anti-OPGLantibody heavy chain constant region (SEQ ID NO: 1) and the amino acidsequence (FIG. 1B) of the anti-OPGL antibody heavy chain constant region(SEQ ID NO: 2).

FIGS. 2A-2B depict a cDNA sequence (FIG. 2A) encoding the anti-OPGLantibody kappa chain constant region (SEQ ID NO: 3) and the amino acidsequence (FIG. 2B) of the anti-OPGL antibody kappa chain constant region(SEQ ID NO: 4).

FIGS. 3A-3B depict a cDNA sequence (FIG. 3A) encoding the 22B3 anti-OPGLantibody heavy chain variable region (SEQ ID NO: 5) and the amino acidsequence (FIG. 3B) of the 22B3 anti-OPGL antibody heavy chain variableregion (SEQ ID NO: 6).

FIGS. 4A-4B depict a cDNA sequence (FIG. 4A) encoding the 22B3 anti-OPGLantibody kappa chain variable region (SEQ ID NO: 7) and the amino acidsequence (FIG. 4B) of the 22B3 anti-OPGL antibody kappa chain variableregion (SEQ ID NO: 8).

FIGS. 5A-5B depict a cDNA sequence (FIG. 5A) encoding the 2E11 anti-OPGLantibody heavy chain variable region (SEQ ID NO: 9) and the amino acidsequence (FIG. 5B) of the 2E11 anti-OPGL antibody heavy chain variableregion (SEQ ID NO: 10).

FIGS. 6A-6B depict a cDNA sequence (FIG. 6A) encoding the 2E11 anti-OPGLantibody kappa chain variable region (SEQ ID NO: 11) and the amino acidsequence (FIG. 6B) of the 2E11 anti-OPGL antibody kappa chain variableregion (SEQ ID NO: 12).

FIGS. 7A-7B depict a cDNA sequence (FIG. 7A) encoding the 2D8 anti-OPGLantibody heavy chain variable region (SEQ ID NO: 13) and the amino acidsequence (FIG. 7B) of the 2D8 anti-OPGL antibody heavy chain variableregion (SEQ ID NO: 14).

FIGS. 8A-8B depict a cDNA sequence (FIG. 8A) encoding the 2D8 anti-OPGLantibody kappa chain variable region (SEQ ID NO: 15) and the amino acidsequence (FIG. 8B) of the 2D8 anti-OPGL antibody kappa chain variableregion (SEQ ID NO: 16).

FIGS. 9A-9B depict a cDNA sequence (FIG. 9A) encoding the 18B2 anti-OPGLantibody heavy chain variable region (SEQ ID NO: 17) and the amino acidsequence (FIG. 9B) of the 18B2 anti-OPGL antibody heavy chain variableregion (SEQ ID NO: 18).

FIGS. 10A-10B depict a cDNA sequence (FIG. 10A) encoding the 18B2anti-OPGL antibody kappa chain variable region (SEQ ID NO: 19) and theamino acid sequence (FIG. 10B) of the 18B2 anti-OPGL antibody kappachain variable region (SEQ ID NO: 20).

FIGS. 11A-11B depict a cDNA sequence (FIG. 11A) encoding the 16E1anti-OPGL antibody heavy chain variable region (SEQ ID NO: 21) and theamino acid sequence (FIG. 11B) of the 16E1 anti-OPGL antibody heavychain variable region (SEQ ID NO: 22).

FIGS. 12A-12B depict a cDNA sequence (FIG. 12A) encoding the 16E1anti-OPGL antibody kappa chain variable region (SEQ ID NO: 23) and theamino acid sequence (FIG. 12B) of the 16E1 anti-OPGL antibody kappachain variable region (SEQ ID NO: 24).

FIGS. 13A-13B depict a cDNA sequence (FIG. 13A) encoding the 9H7anti-OPGL antibody heavy chain variable region (SEQ ID NO: 25) and theamino acid sequence (FIG. 13B) of the 9H7 anti-OPGL antibody heavy chainvariable region (SEQ ID NO: 26).

FIGS. 14A-14B depict a cDNA sequence (FIG. 14A) encoding the 9H7anti-OPGL antibody kappa chain variable region (SEQ ID NO: 27) and theamino acid sequence (FIG. 14B) of the 9H7 anti-OPGL antibody kappa chainvariable region (SEQ ID NO: 28).

FIG. 15 depicts the heavy chain alignment for anti-OPGL antibodiesdesignated 16E1, 2E11, 18B2, 2D8, 22B3, and 9H7. CDRs are underlined,non-consensus amino acids are shaded and in bold type.

FIG. 16 depicts the light chain alignment for anti-OPGL antibodiesdesignated 16E1, 2E11, 18B2, 2D8, 22B3, and 9H7. CDRs are underlined,non-consensus amino acids are shaded and in bold type.

FIG. 17 depicts a circular plasmid map of the pDSRα19:9H7 kappa chainexpression vector.

FIG. 18 shows a circular plasmid map of the pDSRα19:9H7 heavy chainexpression vector.

FIG. 19 depicts an exemplary cell culture process for producinganti-OPGL antibody.

FIG. 20 is a graph showing optical density versus anti-OPGL antibodyconcentration demonstrating OPGL antibody mediated inhibition ofosteoclast formation.

FIG. 21 depicts graphs of serum concentrations of anti-OPGL antibodiesfollowing subcutaneous administration at 1.0 mg/kg in Cynomolgusmonkeys.

FIG. 22 depicts graphs representing the percentage change in serum NTxfrom baseline following subcutaneous administration at 1.0 mg/kg ofanti-OPGL antibodies in Cynomolgus monkeys.

FIG. 23 shows a comparison of murine (SEQ ID NO: 70), human (SEQ ID NO:71), and murine DE variant (SEQ ID NO: 72) amino acid sequences in aregion of OPGL between the D and E regions.

FIG. 24 depicts the results of an enzyme immunoassay showing sixanti-OPGL antibodies of the invention binding murine OPGL (143-317).

FIG. 25 depicts the results of an enzyme immunoassay showing four of theanti-OPGL antibodies of the invention bind FLAG-murine OPGL/DE(158-316).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All references cited in this application are expressly incorporated byreference herein for any purpose.

Definitions

Standard techniques were used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques wereperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures were generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thepresent specification. See e.g., Sambrook et al., 2001, MOLECULARCLONING: A LABORATORY MANUAL, 3d ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., which is incorporated herein byreference for any purpose. Unless specific definitions are provided, thenomenclature utilized in connection with, and the laboratory proceduresand techniques of, analytical chemistry, synthetic organic chemistry,and medicinal and pharmaceutical chemistry described herein are thosewell known and commonly used in the art. Standard techniques can be usedfor chemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

The term “isolated polynucleotide” as used herein means a polynucleotideof genomic, cDNA, or synthetic origin or some combination thereof, whichby virtue of its origin the isolated polynucleotide (1) is notassociated with all or a portion of a polynucleotide in which theisolated polynucleotide is found in nature, (2) is linked to apolynucleotide which it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence.

The term “isolated protein” referred to herein means that a subjectprotein (1) is free of at least some other proteins with which it wouldnormally be found, (2) is essentially free of other proteins from thesame source, e.g., from the same species, (3) is expressed by a cellfrom a different species, (4) has been separated from at least about 50percent of polynucleotides, lipids, carbohydrates, or other materialswith which it is associated in nature, (5) is not associated (bycovalent or noncovalent interaction) with portions of a protein withwhich the “isolated protein” is associated in nature, (6) is operablyassociated (by covalent or noncovalent interaction) with a polypeptidewith which it is not associated in nature, or (7) does not occur innature. Such an isolated protein can be encoded by genomic DNA, cDNA,mRNA or other RNA, of synthetic origin, or any combination thereof.Preferably, the isolated protein is substantially free from proteins orpolypeptides or other contaminants that are found in its naturalenvironment that would interfere with its use (therapeutic, diagnostic,prophylactic, research or otherwise).

The terms “polypeptide” or “protein” means molecules having the sequenceof native proteins, that is, proteins produced by naturally-occurringand specifically non-recombinant cells, or genetically-engineered orrecombinant cells, and comprise molecules having the amino acid sequenceof the native protein, or molecules having deletions from, additions to,and/or substitutions of one or more amino acids of the native sequence.The terms “polypeptide” and “protein” specifically encompass anti-OPGLantibodies, or sequences that have deletions from, additions to, and/orsubstitutions of one or more amino acid of an anti-OPGL antibody.

The term “polypeptide fragment” refers to a polypeptide that has anamino-terminal deletion, a carboxyl-terminal deletion, and/or aninternal deletion. In certain embodiments, fragments are at least 5 toabout 500 amino acids long. It will be appreciated that in certainembodiments, fragments are at least 5, 6, 8, 10, 14, 20, 50, 70, 100,110, 150, 200, 250, 300, 350, 400, or 450 amino acids long. Particularlyuseful polypeptide fragments include functional domains, includingbinding domains. In the case of an anti-OPGL antibody, useful fragmentsinclude but are not limited to a CDR region, a variable domain of aheavy or light chain, a portion of an antibody chain or just itsvariable region including two CDRs, and the like.

The term “immunologically functional immunoglobulin fragment” as usedherein refers to a polypeptide fragment that contains at least the CDRsof the immunoglobulin heavy and light chains. An immunologicallyfunctional immunoglobulin fragment of the invention is capable ofbinding to an antigen. In preferred embodiments, the antigen is a ligandthat specifically binds to a receptor. In these embodiments, binding ofan immunologically functional immunoglobulin fragment of the inventionprevents binding of the ligand to its receptor, interrupting thebiological response resulting from ligand binding to the receptor.Preferably, an immunologically functional immunoglobulin fragment of theinvention binds specifically to OPGL. Most preferably, the fragmentbinds specifically to human OPGL.

The term “naturally-occurring” as used herein and applied to an objectrefers to the fact that the object can be found in nature. For example,a polypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andthat has not been intentionally modified by man is naturally occurring.

The term “operably linked” means that the components to which the termis applied are in a relationship that allows them to carry out theirinherent functions under suitable conditions. For example, a controlsequence “operably linked” to a coding sequence is ligated thereto sothat expression of the protein coding sequence is achieved underconditions compatible with the transcriptional activity of the controlsequences.

The term “control sequence” as used herein refers to polynucleotidesequences that can effect expression, processing or intracellularlocalization of coding sequences to which they are ligated. The natureof such control sequences may differ depending upon the host organism.In particular embodiments, control sequences for prokaryotes may includepromoter, ribosomal binding site, and transcription terminationsequence. In other particular embodiments, control sequences foreukaryotes may include promoters comprising one or a plurality ofrecognition sites for transcription factors, transcription enhancersequences, transcription termination sequences and polyadenylationsequences. In certain embodiments, “control sequences” can includeleader sequences and/or fusion partner sequences.

The term “polynucleotide” as referred to herein means single-stranded ordouble-stranded nucleic acid polymers of at least 10 bases in length. Incertain embodiments, the nucleotides comprising the polynucleotide canbe ribonucleotides or deoxyribonucleotides or a modified form of eithertype of nucleotide. Said modifications include base modifications suchas bromuridine, ribose modifications such as arabinoside and2′,3′-dideoxyribose and internucleotide linkage modifications such asphosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate andphosphoroamidate. The term “polynucleotide” specifically includes singleand double stranded forms of DNA.

The term “oligonucleotide” as used herein includes naturally occurring,and modified nucleotides linked together by naturally occurring, and/ornon-naturally occurring oligonucleotide linkages. Oligonucleotides are apolynucleotide subset comprising members that are generallysingle-stranded and have a length of 200 bases or fewer. In certainembodiments, oligonucleotides are 10 to 60 nucleotides in length. Incertain embodiments, oligonucleotides are 12, 13, 14, 15, 16, 17, 18,19, or 20 to 40 nucleotides in length. Oligonucleotides may be singlestranded or double stranded, e.g. for use in the construction of a genemutant. Oligonucleotides of the invention may be sense or antisenseoligonucleotides with reference to a protein-coding sequence.

The term “naturally occurring nucleotides” includes deoxyribonucleotidesand ribonucleotides. The term “modified nucleotides” includesnucleotides with modified or substituted sugar groups and the like. Theterm “oligonucleotide linkages” includes oligonucleotides linkages suchas phosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoroamidate, and the like. See, e.g., LaPlanche et al., 1986, Nucl.Acids Res. 14: 9081; Stec et al., 1984, J. Am. Chem. Soc. 106: 6077;Stein et al., 1988, Nucl. Acids Res. 16: 3209; Zon et al., 1991,Anti-Cancer Drug Design 6: 539; Zon et al., 1991, OLIGONUCLEOTIDES ANDANALOGUES: A PRACTICAL APPROACH, (F. Eckstein, ed.), Oxford UniversityPress, Oxford England, pp. 87-108; Stec et al., U.S. Pat. No. 5,151,510;Uhlmann and Peyman, 1990, Chemical Reviews 90: 543, the disclosures ofeach of which are hereby incorporated by reference for any purpose. Anoligonucleotide can include a detectable label to enable detection ofthe oligonucleotide or hybridization thereof.

The term “vector” is used to refer to any molecule (e.g., nucleic acid,plasmid, or virus) used to transfer coding information to a host cell.

The term “expression vector” refers to a vector that is suitable fortransformation of a host cell and contains nucleic acid sequences thatdirect and/or control the expression of inserted heterologous nucleicacid sequences. Expression includes, but is not limited to, processessuch as transcription, translation, and RNA splicing, if introns arepresent.

The term “host cell” is used to refer to a cell which has beentransformed, or that is capable of being transformed with a nucleic acidsequence and then of expressing a selected gene of interest. The termincludes the progeny of the parent cell, whether or not the progeny isidentical in morphology or in genetic make-up to the original parent, solong as the selected gene is present.

The term “transduction” is used to refer to the transfer of genes fromone bacterium to another, usually by a phage. “Transduction” also refersto the acquisition and transfer of eukaryotic cellular sequences byretroviruses.

The term “transfection” is used to refer to the uptake of foreign orexogenous DNA by a cell, and a cell has been “transfected” when theexogenous DNA has been introduced inside the cell membrane. A number oftransfection techniques are well known in the art and are disclosedherein. See, e.g., Graham et al., 1973, Virology 52: 456; Sambrook etal., 2001, ibid.; Davis et al., 1986, BASIC METHODS IN MOLECULAR BIOLOGY(Elsevier); and Chu et al., 1981, Gene 13: 197. Such techniques can beused to introduce one or more exogenous DNA moieties into suitable hostcells.

The term “transformation” as used herein refers to a change in a cell'sgenetic characteristics, and a cell has been transformed when it hasbeen modified to contain a new DNA. For example, a cell is transformedwhere it is genetically modified from its native state. Followingtransfection or transduction, the transforming DNA may recombine withthat of the cell by physically integrating into a chromosome of thecell, may be maintained transiently as an episomal element without beingreplicated, or may replicate independently as a plasmid. A cell isconsidered to have been stably transformed when the DNA is replicatedwith the division of the cell.

The term “naturally occurring” or “native” when used in connection withbiological materials such as nucleic acid molecules, polypeptides, hostcells, and the like, refers to materials which are found in nature andare not manipulated by man. Similarly, “non-naturally occurring” or“non-native” as used herein refers to a material that is not found innature or that has been structurally modified or synthesized by man.

The term “antigen” refers to a molecule or a portion of a moleculecapable of being bound by a selective binding agent, such as anantibody, and additionally capable of being used in an animal to produceantibodies capable of binding to an epitope of that antigen. An antigenmay have one or more epitopes.

The term “identity,” as known in the art, refers to a relationshipbetween the sequences of two or more polypeptide molecules or two ormore nucleic acid molecules, as determined by comparing the sequencesthereof. In the art, “identity” also means the degree of sequencerelatedness between nucleic acid molecules or polypeptides, as the casemay be, as determined by the match between two or more nucleotide or twoor more amino acid sequences. “Identity” measures the percent ofidentical matches between the smaller of two or more sequences with gapalignments (if any) addressed by a particular mathematical model orcomputer program (i.e., “algorithms”).

The term “similarity” is used in the art with regard to a relatedconcept, but in contrast to “identity,” “similarity” refers to a measureof relatedness, which includes both identical matches and conservativesubstitution matches. If two polypeptide sequences have, for example,10/20 identical amino acids, and the remainder are all non-conservativesubstitutions, then the percent identity and similarity would both be50%. If in the same example, there are five more positions where thereare conservative substitutions, then the percent identity remains 50%,but the percent similarity would be 75% (15/20). Therefore, in caseswhere there are conservative substitutions, the percent similaritybetween two polypeptides will be higher than the percent identitybetween those two polypeptides.

Identity and similarity of related and polypeptides can be readilycalculated by known methods. Such methods include, but are not limitedto, those described in COMPUTATIONAL MOLECULAR BIOLOGY, (Lesk, A. M.,ed.), 1988, New York: Oxford University Press; BIOCOMPUTING: INFORMATICSAND GENOME PROJECTS, (Smith, D. W., ed.), 1993, New York: AcademicPress; COMPUTER ANALYSIS OF SEQUENCE DATA, PART 1, (Griffin, A. M., andGriffin, H. G., eds.), 1994, New Jersey: Humana Press; von Heinje, G.,1987, SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, New York: Academic Press;SEQUENCE ANALYSIS PRIMER, (Gribskov, M. and Devereux, J., eds.), 1991,New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. AppliedMath. 48:1073; and Durbin et al., 1998, BIOLOGICAL SEQUENCE ANALYSIS,Cambridge University Press.

Preferred methods to determine identity are designed to give the largestmatch between the sequences tested. Methods to determine identity aredescribed in publicly available computer programs. Preferred computerprogram methods to determine identity between two sequences include, butare not limited to, the GCG program package, including GAP (Devereux etal., 1984, Nucl. Acid. Res. 12:387; Genetics Computer Group, Universityof Wisconsin, Madison, Wis., BLASTP, BLASTN, and FASTA, Altschul et al.,1990, J. Mol. Biol. 215: 403-410). The BLASTX program is publiclyavailable from the National Center for Biotechnology Information (NCBI)and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda,Md. 20894; Altschul et al., 1990, supra). The well-known Smith Watermanalgorithm may also be used to determine identity.

Certain alignment schemes for aligning two amino acid sequences mayresult in the matching of only a short region of the two sequences, andthis small aligned region may have very high sequence identity eventhough there is no significant relationship between the two full-lengthsequences. Accordingly, in certain embodiments, the selected alignmentmethod (GAP program) will result in an alignment that spans at least 50contiguous amino acids of the target polypeptide.

For example, using the computer algorithm GAP (Genetics Computer Group,University of Wisconsin, Madison, Wis.), two polypeptides for which thepercent sequence identity is to be determined are aligned for optimalmatching of their respective amino acids (the “matched span”, asdetermined by the algorithm). In certain embodiments, a gap openingpenalty (which is calculated as three times the average diagonal,wherein the “average diagonal” is the average of the diagonal of thecomparison matrix being used; the “diagonal” is the score or numberassigned to each perfect amino acid match by the particular comparisonmatrix) and a gap extension penalty (which is usually one-tenth of thegap opening penalty), as well as a comparison matrix such as PAM 250 orBLOSUM 62 are used in conjunction with the algorithm. In certainembodiments, a standard comparison matrix (see Dayhoff et al., 1978,Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250comparison matrix; Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA 89:10915-10919 for the BLOSUM 62 comparison matrix) is also used by thealgorithm.

In certain embodiments, the parameters for a polypeptide sequencecomparison include the following:

-   -   Algorithm: Needleman et al., 1970, J. Mol. Biol. 48:443-453;    -   Comparison matrix: BLOSUM 62 from Henikoff et al., 1992, supra;    -   Gap Penalty: 12    -   Gap Length Penalty: 4    -   Threshold of Similarity: 0        The GAP program may be useful with the above parameters. In        certain embodiments, the aforementioned parameters are the        default parameters for polypeptide comparisons (along with no        penalty for end gaps) using the GAP algorithm.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See IMMUNOLOGY—A SYNTHESIS, 2ndEdition, (E. S. Golub and D. R. Gren, Eds.), 1991, Sinauer Associates,Sunderland, Mass., which is incorporated herein by reference for anypurpose. Stereoisomers (e.g., D-amino acids) of the twenty conventionalamino acids, unnatural amino acids such as α-, α-disubstituted aminoacids, N-alkyl amino acids, lactic acid, and other unconventional aminoacids may also be suitable components for polypeptides of the presentinvention. Examples of unconventional amino acids include:4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine,ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine,3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and othersimilar amino acids and imino acids (e.g., 4-hydroxyproline). In thepolypeptide notation used herein, the left-hand direction is the aminoterminal direction and the right-hand direction is the carboxy-terminaldirection, in accordance with standard usage and convention.

Similarly, unless specified otherwise, the left-hand end ofsingle-stranded polynucleotide sequences is the 5′ end; the left-handdirection of double-stranded polynucleotide sequences is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to as the transcription direction; sequenceregions on the DNA strand having the same sequence as the RNA and whichare 5′ to the 5′ end of the RNA transcript are referred to as “upstreamsequences”; sequence regions on the DNA strand having the same sequenceas the RNA and which are 3′ to the 3′ end of the RNA transcript arereferred to as “downstream sequences”.

Naturally occurring residues may be divided into classes based on commonside chain properties:

-   -   1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;    -   2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   3) acidic: Asp, Glu;    -   4) basic: His, Lys, Arg;    -   5) residues that influence chain orientation: Gly, Pro; and    -   6) aromatic: Trp, Tyr, Phe.

Conservative amino acid substitutions may encompass non-naturallyoccurring amino acid residues, which are typically incorporated bychemical peptide synthesis rather than by synthesis in biologicalsystems. These include peptidomimetics and other reversed or invertedforms of amino acid moieties.

Non-conservative substitutions may involve the exchange of a member ofone of these classes for a member from another class. Such substitutedresidues may be introduced into regions of the human antibody that arehomologous with non-human antibodies, or into the non-homologous regionsof the molecule.

In making such changes, according to certain embodiments, thehydropathic index of amino acids may be considered. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics. They are: isoleucine (+4.5); valine (+4.2);leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is understood in the art(see, for example, Kyte et al., 1982, J. Mol. Biol. 157:105-131). It isknown that certain amino acids may be substituted for other amino acidshaving a similar hydropathic index or score and still retain a similarbiological activity. In making changes based upon the hydropathic index,in certain embodiments, the substitution of amino acids whosehydropathic indices are within ±2 is included. In certain embodiments,those which are within ±1 are included, and in certain embodiments,those within ±0.5 are included.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biologically functional protein or peptidethereby created is intended for use in immunological embodiments, as inthe present case. In certain embodiments, the greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigen-binding or immunogenicity, i.e., with a biological property ofthe protein.

The following hydrophilicity values have been assigned to these aminoacid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1);glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5);histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5);leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5)and tryptophan (−3.4). In making changes based upon similarhydrophilicity values, in certain embodiments, the substitution of aminoacids whose hydrophilicity values are within ±2 is included, in certainembodiments, those which are within ±1 are included, and in certainembodiments, those within ±0.5 are included. One may also identifyepitopes from primary amino acid sequences on the basis ofhydrophilicity. These regions are also referred to as “epitopic coreregions.”

Exemplary amino acid substitutions are set forth in Table 1.

TABLE 1 Amino Acid Substitutions Original Exemplary Preferred ResiduesSubstitutions Substitutions Ala Val, Leu, Ile Val Arg Lys, Gln, Asn LysAsn Gln Gln Asp Glu Glu Cys Ser, Ala Ser Gln Asn Asn Glu Asp Asp GlyPro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Leu Phe,Norleucine Leu Norleucine, Ile, Ile Val, Met, Ala, Phe Lys Arg, Gln,Asn, Arg 1,4 Diamine-butyric Acid Met Leu, Phe, Ile Leu Phe Leu, Val,Ile, Ala, Leu Tyr Pro Ala Gly Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr,Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe, Leu Ala,Norleucine

A skilled artisan will be able to determine suitable variants of thepolypeptide as set forth herein using well-known techniques. In certainembodiments, one skilled in the art can identify suitable areas of themolecule that can be changed without destroying activity by targetingregions not believed to be important for activity. In other embodiments,the skilled artisan can identify residues and portions of the moleculesthat are conserved among similar polypeptides. In further embodiments,even areas that are important for biological activity or for structuremay be subject to conservative amino acid substitutions withoutdestroying the biological activity or without adversely affecting thepolypeptide structure.

Additionally, one skilled in the art can review structure-functionstudies identifying residues in similar polypeptides that are importantfor activity or structure. In view of such a comparison, the skilledartisan can predict the importance of amino acid residues in a proteinthat correspond to amino acid residues important for activity orstructure in similar proteins. One skilled in the art may opt forchemically similar amino acid substitutions for such predicted importantamino acid residues.

One skilled in the art can also analyze the three-dimensional structureand amino acid sequence in relation to that structure in similarpolypeptides. In view of such information, one skilled in the art maypredict the alignment of amino acid residues of an antibody with respectto its three dimensional structure. In certain embodiments, one skilledin the art may choose not to make radical changes to amino acid residuespredicted to be on the surface of the protein, since such residues maybe involved in important interactions with other molecules. Moreover,one skilled in the art may generate test variants containing a singleamino acid substitution at each desired amino acid residue. The variantscan then be screened using activity assays known to those skilled in theart. Such variants can be used to gather information about suitablevariants. For example, if it was discovered that a change to aparticular amino acid residue resulted in destroyed, undesirablyreduced, or unsuitable activity, variants with such a change can beavoided. In other words, based on information gathered from such routineexperiments, one skilled in the art can readily determine the aminoacids where further substitutions should be avoided either alone or incombination with other mutations.

A number of scientific publications have been devoted to the predictionof secondary structure. See Molt, 1996, Curr. Op. in Biotech. 7:422-427; Chou et al., 1974, Biochemistry 13: 222-245; Chou et al., 1974,Biochemistry 113: 211-222; Chou et al., 1978, Adv. Enzymol. Relat. AreasMol. Biol. 47: 45-148; Chou et al., 1978, Ann. Rev. Biochem. 47: 251-276and Chou et al., 1979, Biophys. J. 26: 367-384. Moreover, computerprograms are currently available to assist with predicting secondarystructure. One method of predicting secondary structure is based uponhomology modeling. For example, two polypeptides or proteins which havea sequence identity of greater than 30%, or similarity greater than 40%often have similar structural topologies. The recent growth of theprotein structural database (PDB) has provided enhanced predictabilityof secondary structure, including the potential number of folds within apolypeptide's or protein's structure. See Holm et al., 1999, Nucl. Acid.Res. 27: 244-247. It has been suggested (Brenner et al., 1997, Curr. Op.Struct. Biol. 7: 369-376) that there are a limited number of folds in agiven polypeptide or protein and that once a critical number ofstructures have been resolved, structural prediction will becomedramatically more accurate.

Additional methods of predicting secondary structure include “threading”(Jones, 1997, Curr. Opin. Struct. Biol. 7: 377-87; Sippl et al., 1996,Structure 4: 15-19), “profile analysis” (Bowie et al., 1991, Science253: 164-170; Gribskov et al., 1990, Meth. Enzym. 183: 146-159; Gribskovet al., 1987, Proc. Nat. Acad. Sci. USA 84: 4355-4358), and“evolutionary linkage” (See Holm, 1999, supra, and Brenner, 1997,supra).

In certain embodiments, antibody variants include glycosylation variantswherein the number and/or type of glycosylation site has been alteredcompared to the amino acid sequences of the parent polypeptide. Incertain embodiments, protein variants comprise a greater or a lessernumber of N-linked glycosylation sites than the native protein. AnN-linked glycosylation site is characterized by the sequence: Asn-X-Seror Asn-X-Thr, wherein the amino acid residue designated as X may be anyamino acid residue except proline. The substitution of amino acidresidues to create this sequence provides a potential new site for theaddition of an N-linked carbohydrate chain. Alternatively, substitutionsthat eliminate or alter this sequence will prevent addition of anN-linked carbohydrate chain present in the native polypeptide. Alsoprovided are rearrangements of N-linked carbohydrate chains wherein oneor more N-linked glycosylation sites (typically those that are naturallyoccurring) are eliminated and one or more new N-linked sites arecreated. Additional preferred antibody variants include cysteinevariants wherein one or more cysteine residues in the parent or nativeamino acid sequence are deleted from or substituted for another aminoacid (e.g., serine). Cysteine variants are useful, inter alia whenantibodies must be refolded into a biologically active conformation, forexample, after the isolation of insoluble inclusion bodies. Cysteinevariants generally have fewer cysteine residues than the native protein,and typically have an even number to minimize interactions resultingfrom unpaired cysteines.

In additional embodiments, antibody variants can include antibodiescomprising a modified Fc fragment or a modified heavy chain constantregion. An Fc fragment, which stands for “fragment that crystallizes,”or a heavy chain constant region can be modified by mutation to conferon an antibody altered binding characteristics. See, for example, Burtonand Woof, 1992, Advances in Immunology 51: 1-84; Ravetch and Bolland,2001, Annu. Rev. Immunol. 19: 275-90; Shields et al., 2001, Journal ofBiol. Chem 276: 6591-6604; Telleman and Junghans, 2000, Immunology 100:245-251; Medesan et al., 1998, Eur. J. Immunol. 28: 2092-2100; all ofwhich are incorporated herein by reference). Such mutations can includesubstitutions, additions, deletions, or any combination thereof, and aretypically produced by site-directed mutagenesis using one or moremutagenic oligonucleotide(s) according to methods described herein, aswell as according to methods known in the art (see, for example,Maniatis et al., MOLECULAR CLONING: A LABORATORY MANUAL, 3rd Ed., 2001,Cold Spring Harbor, N.Y. and Berger and Kimmel, METHODS IN ENZYMOLOGY,Volume 152, Guide to Molecular Cloning Techniques, 1987, Academic Press,Inc., San Diego, Calif., which are incorporated herein by reference).

According to certain embodiments, amino acid substitutions are thosethat: (1) reduce susceptibility to proteolysis, (2) reducesusceptibility to oxidation, (3) alter binding affinity for formingprotein complexes, (4) alter ligand or antigen binding affinities,and/or (5) confer or modify other physicochemical or functionalproperties on such polypeptides. According to certain embodiments,single or multiple amino acid substitutions (in certain embodiments,conservative amino acid substitutions) may be made in thenaturally-occurring sequence (in certain embodiments, in the portion ofthe polypeptide outside the domain(s) forming intermolecular contacts).In certain embodiments, a conservative amino acid substitution typicallydoes not substantially change the structural characteristics of theparent sequence (e.g., a replacement amino acid should not tend to breaka helix that occurs in the parent sequence, or disrupt other types ofsecondary structure that characterizes the parent sequence). Examples ofart-recognized polypeptide secondary and tertiary structures aredescribed in PROTEINS, STRUCTURES AND MOLECULAR PRINCIPLES (Creighton,Ed.), 1984, W. H. New York: Freeman and Company; INTRODUCTION TO PROTEINSTRUCTURE (Branden and Tooze, eds.), 1991, New York: Garland Publishing;and Thornton et at., 1991, Nature 354: 105, each of which areincorporated herein by reference.

Peptide analogs are commonly used in the pharmaceutical industry asnon-peptide drugs with properties analogous to those of the templatepeptide. These types of non-peptide compound are termed “peptidemimetics” or “peptidomimetics”. Fauchere, 1986, Adv. Drug Res. 15: 29;Veber and Freidinger, 1985, TINS p. 392; and Evans et al., 1987, J. Med.Chem. 30: 1229, which are incorporated herein by reference for anypurpose. Such compounds are often developed with the aid of computerizedmolecular modeling. Peptide mimetics that are structurally similar totherapeutically useful peptides may be used to produce a similartherapeutic or prophylactic effect. Generally, peptidomimetics arestructurally similar to a paradigm polypeptide (i.e., a polypeptide thathas a biochemical property or pharmacological activity), such as humanantibody, but have one or more peptide linkages optionally replaced by alinkage selected from: —CH₂NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH— (cis andtrans), —COCH₂—, —CH(OH)CH₂—, and —CH₂SO—, by methods well known in theart. Systematic substitution of one or more amino acids of a consensussequence with a D-amino acid of the same type (e.g., D-lysine in placeof L-lysine) may be used in certain embodiments to generate more stablepeptides. In addition, constrained peptides comprising a consensussequence or a substantially identical consensus sequence variation maybe generated by methods known in the art (Rizo and Gierasch, 1992, Ann.Rev. Biochem. 61: 387), incorporated herein by reference for anypurpose); for example, by adding internal cysteine residues capable offorming intramolecular disulfide bridges which cyclize the peptide.

“Antibody” or “antibody peptide(s)” refer to an intact antibody, or abinding fragment thereof that competes with the intact antibody forspecific binding. In certain embodiments, binding fragments are producedby recombinant DNA techniques. In certain embodiments, binding fragmentsare produced by enzymatic or chemical cleavage of intact antibodies.Binding fragments include, but are not limited to, Fab, Fab′, F(ab′)₂,Fv, and single-chain antibodies.

The term “heavy chain” includes any immunoglobulin polypeptide having aheavy chain constant region and sufficient variable region sequence toconfer specificity for an OPGL. The term “light chain” includes anyimmunoglobulin polypeptide having a light chain constant region andsufficient variable region sequence to confer specificity for an OPGL. Afull-length heavy chain includes a variable region domain, V_(H), andthree constant region domains, C_(H)1, C_(H)2, and C_(H)3. The V_(H)domain is at the amino-terminus of the polypeptide, and the C_(H)3domain is at the carboxyl-terminus The term “heavy chain”, as usedherein, encompasses a full-length heavy chain and fragments thereof. Afull-length light chain includes a variable region domain, V_(L), and aconstant region domain, C_(L). Like the heavy chain, the variable regiondomain of the light chain is at the amino-terminus of the polypeptide.The term “light chain”, as used herein, encompasses a full-length lightchain and fragments thereof. A F(ab) fragment is comprised of one lightchain and the C_(H)1 and variable regions of one heavy chain. The heavychain of a F(ab) molecule cannot form a disulfide bond with anotherheavy chain molecule. A F(ab′) fragment contains one light chain and oneheavy chain that contains more of the constant region, between theC_(H)1 and C_(H)2 domains, such that an interchain disulfide bond can beformed between two heavy chains to form a F(ab′)₂ molecule. The Fvregion comprises the variable regions from both the heavy and lightchains, but lacks the constant regions. Single-chain antibodies are Fvmolecules in which the heavy and light chain variable regions have beenconnected by a flexible linker to form a single polypeptide chain thatforms an antigen-binding region. Single chain antibodies are discussedin detail in WO 88/01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203incorporate by reference.

A bivalent antibody other than a “multispecific” or “multifunctional”antibody, in certain embodiments, is understood to comprise bindingsites having identical antigenic specificity.

In assessing antibody binding and specificity according to theinvention, an antibody substantially inhibits adhesion of a ligand to areceptor when an excess of antibody reduces the quantity of ligand boundto receptor by at least about 20%, 40%, 60%, 80%, 85%, or more (asmeasured in an in vitro competitive binding assay).

The term “epitope” includes any polypeptide determinant, preferably apolypeptide determinant, capable of specific binding to animmunoglobulin or T-cell receptor. In certain embodiments, epitopedeterminants include chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, incertain embodiments, may have specific three dimensional structuralcharacteristics, and/or specific charge characteristics. An epitope is aregion of an antigen that is bound by an antibody. In certainembodiments, an antibody is said to specifically bind an antigen when itpreferentially recognizes its target antigen in a complex mixture ofproteins and/or macromolecules. In certain embodiments, an antibody issaid to specifically bind an antigen when the dissociation constant is≦10⁻⁸ M, in certain embodiments, when the dissociation constant is ≦10⁻⁹M, and in certain embodiments, when the dissociation constant is ≦10⁻¹⁰M.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials.

As used herein, the terms “label” or “labeled” refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotin moieties that can bedetected by labeled avidin (e.g., streptavidin preferably comprising adetectable marker such as a fluorescent marker, a chemiluminescentmarker or an enzymatic activity that can be detected by optical orcolorimetric methods). In certain embodiments, the label can also betherapeutic. Various methods of labeling polypeptides and glycoproteinsare known in the art and may be used advantageously in the methodsdisclosed herein. Examples of labels for polypeptides include, but arenot limited to, the following: radioisotopes or radionuclides (e.g., ³H,¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ^(99m)Tc, ¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent labels(e.g., fluorescein isothiocyanate or FITC, rhodamine, or lanthanidephosphors), enzymatic labels (e.g., horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase), chemiluminescentlabels, hapten labels such as biotinyl groups, and predeterminedpolypeptide epitopes recognized by a secondary reporter (e.g., leucinezipper pair sequences, binding sites for secondary antibodies, metalbinding domains, or epitope tags). In certain embodiments, labels areattached by spacer arms (such as (CH₂)_(n), where n<about 20) of variouslengths to reduce potential steric hindrance.

The term “biological sample”, as used herein, includes, but is notlimited to, any quantity of a substance from a living thing or formerlyliving thing. Such living things include, but are not limited to, mhumans, mice, monkeys, rats, rabbits, and other animals. Such substancesinclude, but are not limited to, blood, serum, urine, cells, organs,tissues, bone, bone marrow, lymph nodes, and skin.

The term “osteopenic disorder” includes, but is not limited to,osteoporosis, osteopenia, Paget's disease, lytic bone metastases,periodontitis, rheumatoid arthritis, and bone loss due toimmobilization. In addition to these bone disorders, certain cancers areknown to increase osteoclast activity and induce bone resorption, suchas breast and prostate cancer and multiple myeloma. These cancers arenow known to produce factors that result in the over-expression of OPGLin the bone, and lead to increased osteoclast numbers and activity.

The term “pharmaceutical agent or drug” as used herein refers to achemical compound or composition capable of inducing a desiredtherapeutic effect when properly administered to a patient.

As used herein, “substantially pure” or “substantially purified” means acompound or species that is the predominant species present (i.e., on amolar basis it is more abundant than any other individual species in thecomposition). In certain embodiments, a substantially purified fractionis a composition wherein the object species comprises at least about 50percent (on a molar basis) of all macromolecular species present. Incertain embodiments, a substantially pure composition will comprise morethan about 80%, 85%, 90%, 95%, or 99% of all macromolar species presentin the composition. In certain embodiments, the species is purified toessential homogeneity (contaminant species cannot be detected in thecomposition by conventional detection methods) wherein the compositionconsists essentially of a single macromolecular species.

The term “patient” includes human and animal subjects.

Unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

The invention provides antibodies, preferably monoclonal antibodies andmost preferably human antibodies, that are immunologically specific forosteoprotegerin ligand (OPGL), a member of the tumor necrosis factor(TNF) family of cytokines that is involved in the formation ofosteoclasts. Increased osteoclast activity correlates with a number ofosteopenic disorders, including post-menopausal osteoporosis, Paget'sdisease, lytic bone metastases, and rheumatoid arthritis. Thus, areduction in OPGL activity may result in a decrease in osteoclastactivity and may reduce the severity of osteopenic disorders. Accordingto certain embodiments of the invention, antibodies directed to OPGL maybe used detect, diagnose, prevent and treat osteopenic disorders,including by not limited to, those mentioned above.

In certain embodiments of the present invention, there is provided afully human monoclonal antibody against human OPGL. In certainembodiments, nucleotide sequences encoding, and amino acid sequencescomprising, heavy and light chain immunoglobulin molecules, particularlysequences corresponding to the variable regions, are provided. Incertain embodiments, sequences corresponding to complementaritydetermining regions (CDR's), specifically from CDR1 through CDR3, areprovided. According to certain embodiments, a hybridoma cell lineexpressing such an immunoglobulin molecule and monoclonal antibody isalso provided. In certain embodiments, the invention provides purifiedhuman monoclonal antibody against human OPGL.

The ability to clone and reconstruct megabase-sized human loci in yeastartificial chromosomes (YACs) and to introduce them into the mousegermline provides an approach to elucidating the functional componentsof very large or crudely mapped loci as well as generating useful modelsof human disease. Furthermore, the utilization of such technology forsubstitution of mouse loci with their human equivalents provides uniqueinsights into the expression and regulation of human gene productsduring development, their communication with other systems, and theirinvolvement in disease induction and progression.

An important practical application of such a strategy is the“humanization” of the mouse humoral immune system. Introduction of humanimmunoglobulin (Ig) loci into mice in which the endogenous Ig genes havebeen inactivated offers the opportunity to study mechanisms underlyingprogrammed expression and assembly of antibodies as well as their rolein B-cell development. Furthermore, such a strategy provides a sourcefor production of fully human monoclonal antibodies (MAbs). Fully humanantibodies are expected to minimize the immunogenic and allergicresponses intrinsic to mouse or mouse-derivatized MAbs, and to therebyincrease the efficacy and safety of the administered antibodies. Fullyhuman antibodies can be used in the treatment of chronic and recurringhuman diseases, such as osteoporosis, inflammation, autoimmunity, andcancer, the treatment thereof requiring repeated antibodyadministration. Thus, one particular advantage of the anti-OPGLantibodies of the invention is that the antibodies are fully human andcan be administered to patients in a non-acute manner while minimizingadverse reactions commonly associated with human anti-mouse antibodiesor other previously described non-fully human antibodies from non-humanspecies.

One skilled in the art can engineer mouse strains deficient in mouseantibody production with large fragments of the human Ig loci so thatthe mice produce human but not mouse antibodies. Large human Igfragments in mouse germline preserve variable gene diversity as well asthe proper regulation of antibody production and expression. Byexploiting the mouse machinery for antibody diversification andselection and the lack of immunological tolerance to human proteins, thereproduced human antibody repertoire in these mouse strains yield highaffinity antibodies against any antigen of interest, including humanantigens. Using hybridoma technology, antigen-specific human mAbs withthe desired specificity can be produced and selected.

In certain embodiments, the skilled artisan can use constant regionsfrom species other than human along with the human variable region(s) toproduce chimeric antibodies.

Naturally Occurring Antibody Structure

Naturally occurring antibody structural units typically comprise atetramer. Each such tetramer typically is composed of two identicalpairs of polypeptide chains, each pair having one full-length “light”chain (in certain embodiments, about 25 kDa) and one full-length “heavy”chain (in certain embodiments, about 50-70 kDa). The amino-terminalportion of each chain typically includes a variable region of about 100to 110 or more amino acids that typically is responsible for antigenrecognition. The carboxy-terminal portion of each chain typicallydefines a constant region that may be responsible for effector function.Human light chains are typically classified as kappa and lambda lightchains. Heavy chains are typically classified as mu, delta, gamma,alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG,IgA, and IgE, respectively. IgG has several subclasses, including, butnot limited to, IgG₁, IgG₂, IgG₃, and IgG₄. IgM has subclassesincluding, but not limited to, IgM₁ and IgM₂. IgA is similarlysubdivided into subclasses including, but not limited to, IgA₁ and IgA₂.Within full-length light and heavy chains, typically, the variable andconstant regions are joined by a “J” region of about 12 or more aminoacids, with the heavy chain also including a “D” region of about 10 moreamino acids. See, e.g., FUNDAMENTAL IMMUNOLOGY, 2nd ed., Ch. 7 (Paul,W., ed.) 1989, New York: Raven Press (incorporated by reference in itsentirety for all purposes). The variable regions of each light/heavychain pair typically form the antigen-binding site.

The variable regions typically exhibit the same general structure ofrelatively conserved framework regions (FR) joined by threehypervariable regions, also called complementarity determining regionsor CDRs. The CDRs from the two chains of each pair typically are alignedby the framework regions, which may enable binding to a specificepitope. From N-terminal to C-terminal, both light and heavy chainvariable regions typically comprise the domains FR1, CDR1, FR2, CDR2,FR3, CDR3 and FR4. The assignment of amino acids to each domain istypically in accordance with the definitions of Kabat Sequences ofProteins of Immunological Interest (1987 and 1991, National Institutesof Health, Bethesda, Md.), or Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342: 878-883.

Bispecific or Bifunctional Antibodies

A bispecific or bifunctional antibody typically is an artificial hybridantibody having two different heavy/light chain pairs and two differentbinding sites. Bispecific antibodies may be produced by a variety ofmethods including, but not limited to, fusion of hybridomas or linkingof Fab′ fragments. See, e.g., Songsivilai & Lachmann, 1990, Clin. Exp.Immunol. 79: 315-321; Kostelny et al., 1992, J. Immunol. 148: 1547-1553.

Preparation of Antibodies

The invention provides antibodies that specifically bind to human OPGL.In certain embodiments, the antibodies can be produced by immunizationwith full-length OPGL or fragments thereof. The antibodies of theinvention can be polyclonal or monoclonal, and/or may be recombinantantibodies. In preferred embodiments, antibodies of the invention arehuman antibodies prepared, for example, by immunization of transgenicanimals capable of producing human antibodies (see, for example, PCTPublished Application No. WO 93/12227).

The complementarity determining regions (CDRs) of the light and heavychain variable regions of anti-OPGL antibody may be grafted to frameworkregions (FRs) from antibodies from the same, or another, species. Incertain embodiments, the CDRs of the light and heavy chain variableregions of anti-OPGL antibody may be grafted to consensus human FRs. Tocreate consensus human FRs, in certain embodiments, FRs from severalhuman heavy chain or light chain amino acid sequences are aligned toidentify a consensus amino acid sequence. In certain embodiments, theFRs of the anti-OPGL antibody heavy chain or light chain are replacedwith the FRs from a different heavy chain or light chain. In certainembodiments, rare amino acids in the FRs of the heavy and light chainsof anti-OPGL antibody are not replaced, while the rest of the FR aminoacids are replaced. Rare amino acids are specific amino acids that arein positions in which they are not usually found in FRs. In certainembodiments, the grafted variable regions from anti-OPGL antibody may beused with a constant region that is different from the constant regionof anti-OPGL antibody. In certain embodiments, the grafted variableregions are part of a single chain Fv antibody. CDR grafting isdescribed, e.g., in U.S. Pat. Nos. 6,180,370, 5,693,762, 5,693,761,5,585,089, and 5,530,101, which are hereby incorporated by reference forany purpose.

Antibodies of the invention are prepared using transgenic mice that havea substantial portion of the human antibody producing locus inserted inantibody-producing cells of the mice, and that are further engineered tobe deficient in producing endogenous, murine, antibodies. Such mice arecapable of producing human immunoglobulin molecules and antibodies anddo not produce or produce substantially reduced amounts of murineimmunoglobulin molecules and antibodies. Technologies utilized forachieving this result are disclosed in the patents, applications, andreferences disclosed in the patents, applications, and referencesdisclosed in the specification herein. In certain embodiments, theskilled worker may employ methods as disclosed in International PatentApplication Publication No. WO 98/24893, which is hereby incorporated byreference for any purpose. See also Mendez et al., 1997, Nature Genetics15: 146-156, which is hereby incorporated by reference for any purpose.

The monoclonal antibodies (mAbs) of the invention can be produced by avariety of techniques, including conventional monoclonal antibodymethodology, e.g., the standard somatic cell hybridization technique ofKohler and Milstein, 1975, Nature 256: 495. Although somatic cellhybridization procedures are preferred, in principle, other techniquesfor producing monoclonal antibodies can be employed, e.g., viral oroncogenic transformation of B-lymphocytes.

The preferred animal system for preparing hybridomas is the mouse.Hybridoma production in the mouse is very well established, andimmunization protocols and techniques for isolation of immunizedsplenocytes for fusion are well known in the art. Fusion partners (e.g.,murine myeloma cells) and fusion procedures are also known.

In a preferred embodiment, human monoclonal antibodies directed againstOPGL can be generated using transgenic mice carrying parts of the humanimmune system rather than the mouse system. These transgenic mice,referred to herein as “HuMab” mice, contain a human immunoglobulin geneminilocus that encodes unrearranged human heavy (μ and γ) and κ lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (Lonberg et al., 1994,Nature 368: 856-859). Accordingly, the mice exhibit reduced expressionof mouse IgM or κ and in response to immunization, the introduced humanheavy and light chain transgenes, undergo class switching and somaticmutation to generate high affinity human IgG κ monoclonal antibodies(Lonberg et al., supra.; Lonberg and Huszar, 1995, Intern. Rev.Immunol., 13: 65-93; Harding and Lonberg, 1995, Ann. N.Y. Acad. Sci 764:536-546). The preparation of HuMab mice is described in detail in Tayloret al., 1992, Nucleic Acids Research, 20: 6287-6295; Chen et al., 1993,International Immunology 5: 647-656; Tuaillon et al., 1994, J. Immunol.152: 2912-2920; Lonberg et al., 1994, Nature 368: 856-859; Lonberg,1994, Handbook of Exp. Pharmacology 113: 49-101; Taylor et al., 1994,International Immunology 6: 579-591; Lonberg and Huszar, 1995, Intern.Rev. Immunol. 13: 65-93; Harding and Lonberg, 1995, Ann. N.Y. Acad. Sci.764: 536-546; Fishwild et al., 1996, Nature Biotechnology 14: 845-851,the contents of all of which are hereby incorporated by reference intheir entirety. See further U.S. Pat. Nos. 5,545,806; 5,569,825;5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318;5,874,299; and 5,770,429; all to Lonberg and Kay, as well as U.S. Pat.No. 5,545,807 to Surani et al.; International Publication Nos. WO93/1227, published Jun. 24, 1993; WO 92/22646, published Dec. 23, 1992;WO 92/03918, published Mar. 19, 1992, the disclosures of all of whichare hereby incorporated by reference in their entirety. Alternatively,the HCo7 and HCo12 transgenic mice strains described in the Examplesbelow can be used to generate human anti-OPGL antibodies.

According to certain embodiments, fully human monoclonal antibodiesspecific for OPGL are produced as follows. Transgenic mice containinghuman immunoglobulin genes are immunized with the antigen of interest.Lymphatic cells (such as B-cells) from the mice that express antibodiesare obtained. Such recovered cells are fused with a myeloid-type cellline to prepare immortal hybridoma cell lines, and such hybridoma celllines are screened and selected to identify hybridoma cell lines thatproduce antibodies specific to the antigen of interest. In certainembodiments, the production of a hybridoma cell line that producesantibodies specific to OPGL is provided.

In certain embodiments of the invention, the antibodies bind to OPGLwith a dissociation constant (K_(d)) of less than 10⁻⁸ M. In certainembodiments, the antibodies of the invention bind to OPGL with a K_(d)of between approximately 10⁻⁸ M and 10⁻¹⁰ M.

In certain embodiments, the antibodies of the invention are of the IgG₁isotype. In certain embodiments of the invention, the antibodiescomprise a human kappa light chain and a human IgG₁ heavy chain. Incertain embodiments, nucleic acid encoding the heavy and light chainscomprising the antibodies of the invention were cloned for expression inmammalian cells. In certain embodiments, the variable regions of theantibodies are ligated to a constant region other than the constantregion for the IgG₁ isotype.

In certain embodiments, conservative modifications to the heavy andlight chains of anti-OPGL antibody (and corresponding modifications tothe encoding nucleic acids) will produce anti-OPGL antibodies havingfunctional and biochemical characteristics similar to those of anti-OPGLantibody. In contrast, substantial modifications in the functionaland/or biochemical characteristics of anti-OPGL antibody may be achievedby creating substitutions in the amino acid sequence of the heavy andlight chains that differ significantly in their effect on maintaining(a) the structure of the molecular backbone in the area of thesubstitution, for example, as a sheet or helical conformation, (b) thecharge or hydrophobicity of the molecule at the target site, or (c) thebulkiness of the side chain.

For example, a “conservative amino acid substitution” may involve asubstitution of a native amino acid residue with a nonnative residuehaving little or no effect on the polarity or charge of the amino acidresidue at that position. Furthermore, any native residue in thepolypeptide may also be substituted with alanine, as has been previouslydescribed for “alanine scanning mutagenesis.”

Desired amino acid substitutions (whether conservative ornon-conservative) can be determined by those skilled in the art at thetime such substitutions are desired. In certain embodiments, amino acidsubstitutions can be used to identify important residues of anti-OPGLantibody, or to increase or decrease the affinity of the anti-OPGLantibodies described herein.

In alternative embodiments, antibodies of the present invention can beexpressed in cell lines other than hybridoma cell lines. In theseembodiments, sequences encoding particular antibodies can be used fortransformation of a suitable mammalian host cell. According to theseembodiments, transformation can be by any known method for introducingpolynucleotides into a host cell, including, for example packaging thepolynucleotide in a virus (or into a viral vector) and transducing ahost cell with the virus (or vector) or by transfection procedures knownin the art, as exemplified by U.S. Pat. Nos. 4,399,216, 4,912,040,4,740,461, and 4,959,455 (which patents are hereby incorporated hereinby reference for any purpose). Generally, the transformation procedureused may depend upon the host to be transformed. Methods forintroduction of heterologous polynucleotides into mammalian cells arewell known in the art and include, but are not limited to,dextran-mediated transfection, calcium phosphate precipitation,polybrene mediated transfection, protoplast fusion, electroporation,encapsulation of the polynucleotide(s) in liposomes, mixing nucleic acidwith positively-charged lipids, and direct microinjection of the DNAinto nuclei.

A nucleic acid molecule encoding the amino acid sequence of a heavychain constant region, a heavy chain variable region, a light chainconstant region, or a light chain variable region of an OPGL antibody ofthe invention is inserted into an appropriate expression vector usingstandard ligation techniques. In a preferred embodiment, the anti-OPGLantibody heavy chain or light chain constant region is appended to theC-terminus of the appropriate variable region and is ligated into anexpression vector. The vector is typically selected to be functional inthe particular host cell employed (i.e., the vector is compatible withthe host cell machinery such that amplification of the gene and/orexpression of the gene can occur). For a review of expression vectors,see METH. ENZ. 185 (Goeddel, ed.), 1990, Academic Press. Typically,expression vectors used in any of the host cells contain sequences forplasmid maintenance and for cloning and expression of exogenousnucleotide sequences. Such sequences, collectively referred to as“flanking sequences” in certain embodiments will typically include oneor more of the following nucleotide sequences: a promoter, one or moreenhancer sequences, an origin of replication, a transcriptionaltermination sequence, a complete intron sequence containing a donor andacceptor splice site, a sequence encoding a leader sequence forpolypeptide secretion, a ribosome binding site, a polyadenylationsequence, a polylinker region for inserting the nucleic acid encodingthe polypeptide to be expressed, and a selectable marker element. Eachof these sequences is discussed below.

Optionally, the vector may contain a “tag”-encoding sequence, i.e., anoligonucleotide molecule located at the 5′ or 3′ end of the OPGLpolypeptide coding sequence; the oligonucleotide sequence encodespolyHis (such as hexaHis), or another “tag” such as FLAG, HA(hemaglutinin influenza virus), or myc, for which commercially availableantibodies exist. This tag is typically fused to the polypeptide uponexpression of the polypeptide, and can serve as a means for affinitypurification or detection of the OPGL antibody from the host cell.Affinity purification can be accomplished, for example, by columnchromatography using antibodies against the tag as an affinity matrix.Optionally, the tag can subsequently be removed from the purified OPGLpolypeptide by various means such as using certain peptidases forcleavage.

Flanking sequences may be homologous (i.e., from the same species and/orstrain as the host cell), heterologous (i.e., from a species other thanthe host cell species or strain), hybrid (i.e., a combination offlanking sequences from more than one source), synthetic or native. Assuch, the source of a flanking sequence may be any prokaryotic oreukaryotic organism, any vertebrate or invertebrate organism, or anyplant, provided that the flanking sequence is functional in, and can beactivated by, the host cell machinery.

Flanking sequences useful in the vectors of this invention may beobtained by any of several methods well known in the art. Typically,flanking sequences useful herein will have been previously identified bymapping and/or by restriction endonuclease digestion and can thus beisolated from the proper tissue source using the appropriate restrictionendonucleases. In some cases, the full nucleotide sequence of a flankingsequence may be known. Here, the flanking sequence may be synthesizedusing the methods described herein for nucleic acid synthesis orcloning.

Whether all or only a portion of the flanking sequence is known, it maybe obtained using polymerase chain reaction (PCR) and/or by screening agenomic library with a suitable probe such as an oligonucleotide and/orflanking sequence fragment from the same or another species. Where theflanking sequence is not known, a fragment of DNA containing a flankingsequence may be isolated from a larger piece of DNA that may contain,for example, a coding sequence or even another gene or genes. Isolationmay be accomplished by restriction endonuclease digestion to produce theproper DNA fragment followed by isolation using agarose gelpurification, Qiagen® column chromatography (Chatsworth, Calif.), orother methods known to the skilled artisan. The selection of suitableenzymes to accomplish this purpose will be readily apparent to one ofordinary skill in the art.

An origin of replication is typically a part of prokaryotic expressionvectors, particularly those purchased commercially, and the origin aidsin the amplification of the vector in a host cell. If the vector ofchoice does not contain an origin of replication site, one may bechemically synthesized based on a known sequence, and ligated into thevector. For example, the origin of replication from the plasmid pBR322(New England Biolabs, Beverly, Mass.) is suitable for most gram-negativebacteria and various origins (e.g., SV40, polyoma, adenovirus, vesicularstomatitus virus (VSV), or papillomaviruses such as HPV or BPV) areuseful for cloning vectors in mammalian cells. Generally, the origin ofreplication component is not needed for mammalian expression vectors(for example, the SV40 origin is often used only because it contains theearly promoter).

A transcription termination sequence is typically located 3′ of the endof a polypeptide-coding region and serves to terminate transcription.Usually, a transcription termination sequence in prokaryotic cells is aG-C rich fragment followed by a poly-T sequence. While the sequence iseasily cloned from a library or even purchased commercially as part of avector, it can also be readily synthesized using methods for nucleicacid synthesis such as those described herein.

A selectable marker gene element encodes a protein necessary for thesurvival and growth of a host cell grown in a selective culture medium.Typical selection marker genes encode proteins that (a) conferresistance to antibiotics or other toxins, e.g., ampicillin,tetracycline, or kanamycin for prokaryotic host cells; (b) complementauxotrophic deficiencies of the cell; or (c) supply critical nutrientsnot available from complex media. Preferred selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, and thetetracycline resistance gene. A bacterial neomycin resistance gene canalso be used for selection in both prokaryotic and eukaryotic hostcells.

Other selection genes can be used to amplify the gene that will beexpressed. Amplification is a process whereby genes that cannot insingle copy be expressed at high enough levels to permit survival andgrowth of cells under certain selection conditions are reiterated intandem within the chromosomes of successive generations of recombinantcells. Examples of suitable amplifiable selectable markers for mammaliancells include dihydrofolate reductase (DHFR) and promoterless thymidinekinase. In the use of these markers mammalian cell transformants areplaced under selection pressure wherein only the transformants areuniquely adapted to survive by virtue of the selection gene present inthe vector. Selection pressure is imposed by culturing the transformedcells under conditions in which the concentration of selection agent inthe medium is successively increased, thereby leading to theamplification of both the selectable gene and the DNA that encodesanother gene, such as an antibody that binds to OPGL polypeptide. As aresult, increased quantities of a polypeptide such as an anti-OPGLantibody are synthesized from the amplified DNA. A ribosome-binding siteis usually necessary for translation initiation of mRNA and ischaracterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozaksequence (eukaryotes). The element is typically located 3′ to thepromoter and 5′ to the coding sequence of the polypeptide to beexpressed.

In some cases, for example where glycosylation is desired in aeukaryotic host cell expression system, various presequences can bemanipulated to improve glycosylation or yield. For example, thepeptidase cleavage site of a particular signal peptide can be altered,or pro-sequences added, which also may affect glycosylation. The finalprotein product may have, in the −1 position (relative to the firstamino acid of the mature protein) one or more additional amino acidsincident to expression, which may not have been totally removed. Forexample, the final protein product may have one or two amino acidresidues found in the peptidase cleavage site, attached to theamino-terminus. Alternatively, use of some enzyme cleavage sites mayresult in a slightly truncated yet active form of the desiredpolypeptide, if the enzyme cuts at such area within the maturepolypeptide.

The expression and cloning vectors of the present invention willtypically contain a promoter that is recognized by the host organism andoperably linked to nucleic acid encoding the anti-OPGL antibody.Promoters are untranscribed sequences located upstream (i.e., 5′) to thestart codon of a structural gene (generally within about 100 to 1000 bp)that control transcription of the structural gene. Promoters areconventionally grouped into one of two classes: inducible promoters andconstitutive promoters. Inducible promoters initiate increased levels oftranscription from DNA under their control in response to some change inculture conditions, such as the presence or absence of a nutrient or achange in temperature. Constitutive promoters, on the other hand,initiate continual gene product production; that is, there is little orno experimental control over gene expression. A large number ofpromoters, recognized by a variety of potential host cells, are wellknown. A suitable promoter is operably linked to the DNA encodinganti-OPGL antibody by removing the promoter from the source DNA byrestriction enzyme digestion or amplifying the promoter by polymerasechain reaction and inserting the desired promoter sequence into thevector.

Suitable promoters for use with yeast hosts are also well known in theart. Yeast enhancers are advantageously used with yeast promoters.Suitable promoters for use with mammalian host cells are well known andinclude, but are not limited to, those obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, retroviruses, hepatitis-B virus and most preferablySimian Virus 40 (SV40). Other suitable mammalian promoters includeheterologous mammalian promoters, for example, heat-shock promoters andthe actin promoter.

Additional promoters that may be of interest include, but are notlimited to: the SV40 early promoter region (Bernoist and Chambon, 1981,Nature 290: 304-10); the CMV promoter; the promoter contained in the 3′long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell22: 787-97); the herpes thymidine kinase promoter (Wagner et al., 1981,Proc. Natl. Acad. Sci. U.S.A. 78: 1444-45); the regulatory sequences ofthe metallothionine gene (Brinster et al., 1982, Nature 296: 39-42);prokaryotic expression vectors such as the beta-lactamase promoter(Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. U.S.A., 75:3727-31); or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad.Sci. U.S.A. 80: 21-25). Also of interest are the following animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgenic animals: the elastase I gene controlregion that is active in pancreatic acinar cells (Swift et al., 1984,Cell 38: 639-46; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant.Biol. 50: 399-409; MacDonald, 1987, Hepatology 7: 425-515); the insulingene control region that is active in pancreatic beta cells (Hanahan,1985, Nature 315: 115-22); the mouse mammary tumor virus control regionthat is active in testicular, breast, lymphoid and mast cells (Leder etal., 1986, Cell 45: 485-95); the albumin gene control region that isactive in liver (Pinkert et al., 1987, Genes and Devel. 1: 268-76); thealpha-feto-protein gene control region that is active in liver (Krumlaufet al., 1985, Mol. Cell. Biol. 5: 1639-48; Hammer et al., 1987, Science235: 53-58); the alpha 1-antitrypsin gene control region that is activein the liver (Kelsey et al., 1987, Genes and Devel. 1: 161-71); thebeta-globin gene control region that is active in myeloid cells (Mogramet al., 1985, Nature 315: 338-40; Kollias et al., 1986, Cell 46: 89-94);the myelin basic protein gene control region that is active inoligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-12); the myosin light chain-2 gene control region that is active inskeletal muscle (Sani, 1985, Nature 314: 283-86); the gonadotropicreleasing hormone gene control region that is active in the hypothalamus(Mason et al., 1986, Science 234: 1372-78); and most particularly theimmunoglobulin gene control region that is active in lymphoid cells(Grosschedl et al., 1984, Cell 38: 647-58; Adames et al., 1985, Nature318: 533-38; Alexander et al., 1987, Mol. Cell Biol. 7: 1436-44).

An enhancer sequence may be inserted into the vector to increase thetranscription of a nucleic acid encoding an anti-OPGL antibody of thepresent invention by higher eukaryotes. Enhancers are cis-actingelements of DNA, usually about 10-300 bp in length, that act onpromoters to increase transcription. Enhancers are relativelyorientation and position independent. They have been found 5′ and 3′ tothe transcription unit. Several enhancer sequences available frommammalian genes are known (e.g., globin, elastase, albumin,alpha-feto-protein and insulin). Typically, however, an enhancer from avirus will be used. The SV40 enhancer, the cytomegalovirus earlypromoter enhancer, the polyoma enhancer, and adenovirus enhancers areexemplary enhancing elements for the activation of eukaryotic promoters.While an enhancer may be spliced into the vector at a position 5′ or 3′to a nucleic acid molecule, it is typically located at a site 5′ fromthe promoter.

Expression vectors of the invention may be constructed from a convenientstarting vector such as a commercially available vector. Such vectorsmay or may not contain all of the desired flanking sequences. Where oneor more of the flanking sequences described herein are not alreadypresent in the vector, they may be individually obtained and ligatedinto the vector. Methods used for obtaining each of the flankingsequences are well known to one skilled in the art.

After the vector has been constructed and a nucleic acid moleculeencoding an anti-OPGL antibody has been inserted into the proper site ofthe vector, the completed vector may be inserted into a suitable hostcell for amplification and/or polypeptide expression. The transformationof an expression vector for an anti-OPGL antibody into a selected hostcell may be accomplished by well-known methods including methods such astransfection, infection, calcium chloride, electroporation,microinjection, lipofection, DEAE-dextran method, or other knowntechniques. The method selected will in part be a function of the typeof host cell to be used. These methods and other suitable methods arewell known to the skilled artisan, and are set forth, for example, inSambrook et al., supra.

A host cell, when cultured under appropriate conditions, synthesizes ananti-OPGL antibody that can subsequently be collected from the culturemedium (if the host cell secretes it into the medium) or directly fromthe host cell producing it (if it is not secreted). The selection of anappropriate host cell will depend upon various factors, such as desiredexpression levels, polypeptide modifications that are desirable ornecessary for activity (such as glycosylation or phosphorylation) andease of folding into a biologically active molecule.

Mammalian cell lines available as hosts for expression are well known inthe art and include, but are not limited to, many immortalized celllines available from the American Type Culture Collection (ATCC), suchas Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney(BHK) cells, monkey kidney cells (COS), human hepatocellular carcinomacells (e.g., Hep G2), and a number of other cell lines. In certainembodiments, cell lines may be selected through determining which celllines have high expression levels and produce antibodies withconstitutive OPGL binding properties. In another embodiment, a cell linefrom the B cell lineage that does not make its own antibody but has acapacity to make and secrete a heterologous antibody can be selected.

Antibodies of the invention are useful for detecting OPGL in biologicalsamples and identification of cells or tissues that produce the protein.In certain embodiments, antibodies that bind to OPGL and blockinteraction with other binding compounds may have therapeutic use inmodulating osteoclast differentiation and bone resorption. In certainembodiments, antibodies to OPGL may block OPGL binding to ODAR (RANK),which may result in a block in the signal transduction cascade and lossof NF-kB mediated transcription activation. Assays for measuringNF-kB-mediated transcription activation using, e.g., a luciferasereporter assay, are known to those skilled in the art.

In certain embodiments, antibodies to OPGL may be useful in treatment ofbone diseases such as osteoporosis and Paget's disease. In certainembodiments, antibodies can be tested for binding to OPGL in the absenceor presence of OPG and examined for their ability to inhibitOPGL-mediated osteoclastogenesis and/or bone resorption.

Anti-OPGL antibodies of the invention can be administered alone or incombination with other therapeutic agents, in particular, in combinationwith other cancer therapy agents. Such agents generally includeradiation therapy or chemotherapy. Chemotherapy, for example, caninvolve treatment with one or more of the following: anthracyclines,taxol, tamoxifene, doxorubicin, 5-fluorouracil, and other drugs known tothe skilled worker.

In addition, anti-OPGL antibodies can be administered to patients incombination with antibodies that bind to tumor cells and induce acytotoxic and/or cytostatic effect on tumor growth. Examples of suchantibodies include those that bind to cell surface proteins Her2, CDC20,CDC33, mucin-like glycoprotein and epidermal growth factor receptor(EGFR) present on tumor cells and induce a cytostatic and/or cytotoxiceffect on tumor cells displaying these proteins. Examples of suchantibodies include HERCEPTIN for treatment of breast cancer and RITUXANfor the treatment of non-Hodgkin's lymphoma. Also, combination therapycan include as cancer therapy agents polypeptides that selectivelyinduce apoptosis in tumor cells, such as the TNF-related polypeptideTRAIL. Anti-OPGL or antigen binding fragments of the invention can beadministered prior to, concurrent with, or subsequent to treatment witha cancer therapy agent. Anti-OPGL antibodies can be administeredprophylactically to prevent or mitigate the onset of loss of bone massby metastatic cancer or can be given for the treatment of an existingcondition of loss of bone mass due to metastasis.

Anti-OPGL antibodies of the invention may be used to prevent and/ortreat the growth of tumor cells in bone. Cancer that metastasizes tobone can spread readily as tumor cells stimulate osteoclasts to resorbthe internal bone matrix. Treatment with an anti-OPGL antibody willmaintain bone density by inhibiting resorption and decrease thelikelihood of tumor cells spreading throughout the skeleton. Any cancerthat metastasizes to bone may be prevented and/or treated with ananti-OPGL antibody.

In one embodiment, multiple myeloma may be prevented and/or treated withan anti-OPGL antibody or antigen binding fragment thereof. Multiplemyeloma is localized to bone. Affected patients typically exhibit a lossof bone mass due to increased osteoclast activation in localizedregions. Myeloma cells either directly or indirectly produce OPGL, whichin turn activates osteoclasts resulting in local bone lysis surroundingthe myeloma cells embedded in bone marrow spaces. The normal osteoclastsadjacent to the myeloma cell in turn produce IL-6, leading to growth andproliferation of myeloma cells. Myeloma cells expand in a clonal fashionand occupy bone spaces that are being created by inappropriate boneresorption. Treatment of an animal with an anti-OPGL antibody blocksactivation of osteoclasts which in turn leads to a decrease in IL-6production by osteoclasts, and a suppression of myeloma all growthand/or proliferation.

Anti-OPGL antibodies may be used alone for the treatment of the abovereferenced conditions resulting in loss of bone mass or in combinationwith a therapeutically effective amount of a bone growth promoting(anabolic) agent or a bone anti-resorptive agent including but notlimited to bone morphogenic factors designated BMP-1 to BMP-12,transforming growth factor-β and TGF-β family members, fibroblast growthfactors FGF-1 to FGF-10, interleukin-1 inhibitors, TNFα inhibitors,parathyroid hormone, E series prostaglandins, bisphosphonates andbone-enhancing minerals such as fluoride and calcium. Anabolic agentsinclude parathyroid hormone and insulin-like growth factor (IGF),wherein the latter agent is preferably complexed with an IGF bindingprotein. Preferred embodiments also include the combination of ananti-OPGL antibody with an interluekin-1 (IL-1) receptor antagonist oran anti-OPGL antibody with a soluble TNF receptor, such as soluble TNFreceptor-1 or soluble TNF receptor-2. An exemplary IL-1 receptorantagonist is described in WO89/11540 and an exemplary soluble TNFreceptor-1 is described in WO98/01555.

In preferred embodiments, the invention provides pharmaceuticalcompositions comprising a therapeutically effective amount of theantibodies of the invention together with a pharmaceutically acceptablediluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant.In certain embodiments, pharmaceutical compositions comprising atherapeutically effective amount of anti-OPGL antibodies are provided.

Acceptable formulation materials preferably are nontoxic to recipientsat the dosages and concentrations employed. The pharmaceuticalcomposition may contain formulation materials for modifying, maintainingor preserving, for example, the pH, osmolarity, viscosity, clarity,color, isotonicity, odor, sterility, stability, rate of dissolution orrelease, adsorption or penetration of the composition. Suitableformulation materials include, but are not limited to, amino acids (suchas glycine, glutamine, asparagine, arginine or lysine); antimicrobials;antioxidants (such as ascorbic acid, sodium sulfite or sodiumhydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl,citrates, phosphates or other organic acids); bulking agents (such asmannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides; and other carbohydrates (such as glucose, mannose ordextrins); proteins (such as serum albumin, gelatin or immunoglobulins);coloring, flavoring and diluting agents; emulsifying agents; hydrophilicpolymers (such as polyvinylpyrrolidone); low molecular weightpolypeptides; salt-forming counterions (such as sodium); preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such asglycerin, propylene glycol or polyethylene glycol); sugar alcohols (suchas mannitol or sorbitol); suspending agents; surfactants or wettingagents (such as pluronics, PEG, sorbitan esters, polysorbates such aspolysorbate 20, polysorbate 80, triton, tromethamine, lecithin,cholesterol, tyloxapal); stability enhancing agents (such as sucrose orsorbitol); tonicity enhancing agents (such as alkali metal halides,preferably sodium or potassium chloride, mannitol sorbitol); deliveryvehicles; diluents; excipients and/or pharmaceutical adjuvants.(REMINGTON'S PHARMACEUTICAL SCIENCES, 18^(th) Edition, (A. R. Gennaro,ed.), 1990, Mack Publishing Company.

In certain embodiments, optimal pharmaceutical compositions will bedetermined by one skilled in the art depending upon, for example, theintended route of administration, delivery format and desired dosage.See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, ibid. Suchcompositions may influence the physical state, stability, rate of invivo release and rate of in vivo clearance of the antibodies of theinvention.

The primary vehicle or carrier in a pharmaceutical composition may beeither aqueous or non-aqueous in nature. For example, a suitable vehicleor carrier may be water for injection, physiological saline solution orartificial cerebrospinal fluid, possibly supplemented with othermaterials common in compositions for parenteral administration. Neutralbuffered saline or saline mixed with serum albumin are further exemplaryvehicles. Pharmaceutical compositions can comprise Tris buffer of aboutpH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may furtherinclude sorbitol or a suitable substitute therefor. Anti-OPGL antibodycompositions may be prepared for storage by mixing the selectedcomposition having the desired degree of purity with optionalformulation agents (REMINGTON'S PHARMACEUTICAL SCIENCES, ibid.) in theform of a lyophilized cake or an aqueous solution. Further, theanti-OPGL antibody product may be formulated as a lyophilizate usingappropriate excipients such as sucrose.

Formulation components are present in concentrations that are acceptableto the site of administration. Buffers are advantageously used tomaintain the composition at physiological pH or at a slightly lower pH,typically within a pH range of from about 5 to about 8.

The pharmaceutical compositions of the invention can be deliveredparenterally. When parenteral administration is contemplated, thetherapeutic compositions for use in this invention may be in the form ofa pyrogen-free, parenterally acceptable aqueous solution comprising thedesired anti-OPGL antibody in a pharmaceutically acceptable vehicle. Aparticularly suitable vehicle for parenteral injection is steriledistilled water in which the anti-OPGL antibody is formulated as asterile, isotonic solution, properly preserved. Preparation can involvethe formulation of the desired molecule with an agent, such asinjectable microspheres, bio-erodible particles, polymeric compounds(such as polylactic acid or polyglycolic acid), beads or liposomes, thatmay provide controlled or sustained release of the product which maythen be delivered via a depot injection. Formulation with hyaluronicacid has the effect of promoting sustained duration in the circulation.Implantable drug delivery devices may be used to introduce the desiredmolecule.

The compositions may be selected for inhalation. In these embodiments,an anti-OPGL antibody is formulated as a dry powder for inhalation, oranti-OPGL antibody inhalation solutions may also be formulated with apropellant for aerosol delivery, such as by nebulization. Pulmonaryadministration is further described in PCT Application No.PCT/US94/001875, which describes pulmonary delivery of chemicallymodified proteins.

The pharmaceutical compositions of the invention can be deliveredthrough the digestive tract, such as orally. The preparation of suchpharmaceutically acceptable compositions is within the skill of the art.Anti-OPGL antibodies that are administered in this fashion may beformulated with or without those carriers customarily used in thecompounding of solid dosage forms such as tablets and capsules. Acapsule may be designed to release the active portion of the formulationat the point in the gastrointestinal tract when bioavailability ismaximized and pre-systemic degradation is minimized Additional agentscan be included to facilitate absorption of the anti-OPGL antibody.Diluents, flavorings, low melting point waxes, vegetable oils,lubricants, suspending agents, tablet disintegrating agents, and bindersmay also be employed.

A pharmaceutical composition may involve an effective quantity ofanti-OPGL antibodies in a mixture with non-toxic excipients that aresuitable for the manufacture of tablets. By dissolving the tablets insterile water, or another appropriate vehicle, solutions may be preparedin unit-dose form. Suitable excipients include, but are not limited to,inert diluents, such as calcium carbonate, sodium carbonate orbicarbonate, lactose, or calcium phosphate; or binding agents, such asstarch, gelatin, or acacia; or lubricating agents such as magnesiumstearate, stearic acid, or talc.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving anti-OPGL antibodies insustained- or controlled-delivery formulations. Techniques forformulating a variety of other sustained- or controlled-delivery means,such as liposome carriers, bio-erodible microparticles or porous beadsand depot injections, are also known to those skilled in the art. See,for example, PCT Application No. PCT/US93/00829, which describes thecontrolled release of porous polymeric microparticles for the deliveryof pharmaceutical compositions. Sustained-release preparations mayinclude semipermeable polymer matrices in the form of shaped articles,e.g. films, or microcapsules, polyesters, hydrogels, polylactides (U.S.Pat. No. 3,773,919 and EP 058,481), copolymers of L-glutamic acid andgamma ethyl-L-glutamate (Sidman et al., 1983, Biopolymers 22: 547-556),poly(2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed.Mater. Res. 15: 167-277) and Langer, 1982, Chem. Tech. 12: 98-105),ethylene vinyl acetate (Langer et al., ibid.) orpoly-D(−)-3-hydroxybutyric acid (EP 133,988). Sustained releasecompositions may also include liposomes, which can be prepared by any ofseveral methods known in the art. See e.g., Eppstein et al., 1985, Proc.Natl. Acad. Sci. USA 82: 3688-3692; EP 036,676; EP 088,046 and EP143,949.

The pharmaceutical composition to be used for in vivo administrationtypically is sterile. In certain embodiments, this may be accomplishedby filtration through sterile filtration membranes. In certainembodiments, where the composition is lyophilized, sterilization usingthis method may be conducted either prior to or following lyophilizationand reconstitution. In certain embodiments, the composition forparenteral administration may be stored in lyophilized form or in asolution. In certain embodiments, parenteral compositions generally areplaced into a container having a sterile access port, for example, anintravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

Once the pharmaceutical composition of the invention has beenformulated, it may be stored in sterile vials as a solution, suspension,gel, emulsion, solid, or as a dehydrated or lyophilized powder. Suchformulations may be stored either in a ready-to-use form or in a form(e.g., lyophilized) that is reconstituted prior to administration.

The invention also provides kits for producing a single-doseadministration unit. The kits of the invention may each contain both afirst container having a dried protein and a second container having anaqueous formulation, including for example single and multi-chamberedpre-filled syringes (e.g., liquid syringes, lyosyringes or needle-freesyringes).

The effective amount of an anti-OPGL antibody pharmaceutical compositionto be employed therapeutically will depend, for example, upon thetherapeutic context and objectives. One skilled in the art willappreciate that the appropriate dosage levels for treatment, accordingto certain embodiments, will thus vary depending, in part, upon themolecule delivered, the indication for which the anti-OPGL antibody isbeing used, the route of administration, and the size (body weight, bodysurface or organ size) and/or condition (the age and general health) ofthe patient. In certain embodiments, the clinician may titer the dosageand modify the route of administration to obtain the optimal therapeuticeffect. Typical dosages range from about 0.1 μg/kg to up to about 30mg/kg or more, depending on the factors mentioned above. In certainembodiments, the dosage may range from 0.1 μg/kg up to about 30 mg/kg;or 1 μg/kg up to about 30 mg/kg; or 5 μg/kg up to about 30 mg/kg.

Dosing frequency will depend upon the pharmacokinetic parameters of theanti-OPGL antibody in the formulation used. For example, a clinicianwill administer the composition until a dosage is reached that achievesthe desired effect. The composition may therefore be administered as asingle dose, or as two or more doses (which may or may not contain thesame amount of the desired molecule) over time, or as a continuousinfusion via an implantation device or catheter. Further refinement ofthe appropriate dosage is routinely made by those of ordinary skill inthe art and is within the ambit of tasks routinely performed by them.Appropriate dosages may be ascertained through use of appropriatedose-response data.

Administration routes for the pharmaceutical compositions of theinvention include orally, through injection by intravenous,intraperitoneal, intracerebral (intra-parenchymal),intracerebroventricular, intramuscular, intra-ocular, intraarterial,intraportal, or intralesional routes; by sustained release systems or byimplantation devices. The pharmaceutical compositions may beadministered by bolus injection or continuously by infusion, or byimplantation device. The pharmaceutical composition also can beadministered locally via implantation of a membrane, sponge or anotherappropriate material onto which the desired molecule has been absorbedor encapsulated. Where an implantation device is used, the device may beimplanted into any suitable tissue or organ, and delivery of the desiredmolecule may be via diffusion, timed-release bolus, or continuousadministration.

It also may be desirable to use anti-OPGL antibody pharmaceuticalcompositions according to the invention ex vivo. In such instances,cells, tissues or organs that have been removed from the patient areexposed to anti-OPGL antibody pharmaceutical compositions after whichthe cells, tissues and/or organs are subsequently implanted back intothe patient.

In particular, anti-OPGL antibody can be delivered by implanting certaincells that have been genetically engineered, using methods such as thosedescribed herein, to express and secrete the polypeptide. In certainembodiments, such cells may be animal or human cells, and may beautologous, heterologous, or xenogeneic, or may be immortalized. Incertain embodiments, the cells may be immortalized. In otherembodiments, in order to decrease the chance of an immunologicalresponse, the cells may be encapsulated to avoid infiltration ofsurrounding tissues. In further embodiments, the encapsulation materialsare typically biocompatible, semi-permeable polymeric enclosures ormembranes that allow the release of the protein product(s) but preventthe destruction of the cells by the patient's immune system or by otherdetrimental factors from the surrounding tissues.

Examples

The following examples, including the experiments conducted and resultsachieved are provided for illustrative purposes only and are not to beconstrued as limiting the present invention.

Example 1 Production of Human Monoclonal Antibodies Against OPGLTransgenic HuMab Mice

Fully human monoclonal antibodies to OPGL were prepared using HCo7,HCo12, and HCo7+HCo12 strains of transgenic mice, each of whichexpresses human antibody genes. In each of these mouse strains, theendogenous mouse kappa light chain gene has been homozygously disrupted(as described in Chen et al., 1993, EMBO J. 12: 811-820) and theendogenous mouse heavy chain gene has been homozygously disrupted asdescribed in Example 1 of PCT Publication WO 01/09187 (incorporated byreference). Each of these mouse strains carries a human kappa lightchain transgene, KCo5 (as described in Fishwild et al., 1996, NatureBiotechnology 14: 845-851). The HCo7 strain carries the HCo7 human heavychain transgene as described in U.S. Pat. Nos. 5,545,806; 5,625,825; and5,545,807 (incorporated by reference). The HCo12 strain carries theHCo12 human heavy chain transgene as described in Example 2 of PCTPublication WO 01/09187 (incorporated by reference). The HCo7+HCo12strain carries both the HCo7 and the HCo12 heavy chain transgenes and ishemizygous for each transgene. All of these strains are referred toherein as HuMab mice.

HuMab Immunizations:

To generate fully human monoclonal antibodies to OPGL, HuMab mice wereimmunized with purified recombinant OPGL derived from E. coli or CHOcells as antigen. General immunization schemes for HuMab mice aredescribed in Lonberg et al. 91994, Nature 368: 856-859, Fishwild et al.,ibid., and PCT Publication WO 98/24884 (the teachings of each of whichare incorporated by reference). Mice were 6-16 weeks of age upon thefirst infusion of antigen. A purified recombinant preparation (50-100μg) of OPGL antigen (e.g., purified from transfected E. coli or CHOcells expressing OPGL) was used to immunize the HuMab miceintraperitoneally (IP) or subcutaneously (Sc).

Immunizations of HuMab transgenic mice were performed twice usingantigen in complete Freund's adjuvant, followed by 2-4 weeks IPimmunization (up to a total of 9 immunizations) with the antigen inincomplete Freund's adjuvant. Several dozen mice were immunized for eachantigen. A total of 136 HuMab mice of the HCo7, HCo12, and HCo7+HCo12strains were immunized with OPGL. The immune response was monitored byretroorbital bleeds.

To select HuMab mice producing antibodies that bound OPGL, sera fromimmunized mice was tested by ELISA as described by Fishwild et al.supra. Briefly, microtiter plates were coated with purified recombinantOPGL from CHO cells or E. coli at 1-2 μL/mL in PBS and 50 μL/wellincubated at 4° C. overnight, then blocked with 200 μL/well with 5%chicken serum in PBS/Tween (0.05%). Dilutions of plasma fromOPGL-immunized mice were added to each well and incubated for 1-2 hoursat ambient temperature. The plates were washed with PBS/Tween and thenincubated with a goat anti-human IgG Fc-specific polyclonal reagentconjugated to horseradish peroxidase (HRP) for 1 hour at roomtemperature. After washing, the plates were developed with ABTSsubstrate (Sigma, A-1888, 0.22 mg/mL) and analyzed at OD of 415-495.Mice with sufficient titers of anti-OPGL human immunoglobulin were usedto produce monoclonal antibodies as described below.

Generation of Hybridomas Producing Human Monoclonal Antibodies to OPGL

Mice were prepared for monoclonal antibody production by boosting withantigen intravenously 2 days before sacrifice, and spleens were removedthereafter. The mouse splenocytes were isolated from the HuMab mice andfused with PEG to a mouse myeloma cell line based upon standardprotocols. Typically, 10-20 fusions for each antigen were performed.

Briefly, single cell suspensions of splenic lymphocytes from immunizedmice were fused to one-quarter the number of P3X63-Ag8.653 nonsecretingmouse myeloma cells (ATCC, CRL 1580) using 50% PEG (Sigma Chemical Co.,St. Louis, Mo.). Cells were plated at approximately 1×10⁵ cells/well inflat bottom microtitre plates, followed by about two week incubation inselective medium containing 10% fetal bovine serum, 10% P388D1 (ATCC,CRL TIB-63) conditioned medium and 3-5% origen (IGEN) in DMEM(Mediatech, CRL 10013, with high glucose, L-glutamine and sodiumpyruvate) plus 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 mg/mLgentamycin and 1× HAT (Sigma, CRL P-7185). After 1-2 weeks, cells werecultured in medium in which the HAT was replaced with HT.

The resulting hybridomas were screened for the production ofantigen-specific antibodies. Individual wells were screened by ELISA(described above) for human anti-OPGL monoclonal IgG antibodies. Onceextensive hybridoma growth occurred, medium was monitored usually after10-14 days. Antibody secreting hybridomas were replated, screened againand, if still positive by ELISA for human IgG, anti-OPGL monoclonalantibodies were subcloned at least twice by limiting dilution. Thestable subclones were then cultured in vitro to generate small amountsof antibody in tissue culture medium for characterization.

Selection of Human Monoclonal Antibodies that Bind to OPGL

An ELISA assay as described above was used to screen for hybridomas thatshowed positive reactivity with OPGL immunogen. Hybridomas secreting amonoclonal antibody that bound with high avidity to OPGL were subclonedand further characterized. One clone from each hybridoma, which retainedthe reactivity of parent cells (as determined by ELISA), was chosen formaking a 5-10 vial cell bank stored in liquid nitrogen.

An isotype-specific ELISA was performed to determine the isotype of themonoclonal antibodies produced as disclosed herein. In theseexperiments, microtitre plate wells were coated with 50 μL/well of asolution of 1 μg/mL mouse anti-human kappa light chain in PBS andincubated at 4° C. overnight. After blocking with 5% chicken serum, theplates were reacted with supernatant from each tested monoclonalantibody and a purified isotype control. Plates were incubated atambient temperature for 1-2 hours. The wells were then reacted witheither human IgG₁ or IgG₃-specific horseradish peroxidase-conjugatedgoat anti-human polyclonal antisera and plates developed and analyzed asdescribed above.

Monoclonal antibodies purified from six hybridoma supernatants thatshowed significant binding to OPGL as detected by ELISA were furthertested for biological activity using in vitro receptor binding assaysand human OPGL-dependent in vitro osteoclast forming assays (describedin Example 6 below). The antibodies selected were designated 16E1, 2E11,18B2, 2D8, 22B3, and 9H7. The heavy chain alignment for these anti-OPGLantibodies is shown in FIG. 15. The light chain alignment for theanti-OPGL antibodies is shown in FIG. 16. Non-consensus sequences areshown in bold and are shaded, and complementarity-determining regions(CDRs) are underlined.

Example 2 Cloning the 9H7 Anti-OPGL Heavy and Light Chains Cloning ofthe 9H7 Anti-OPGL MAb Light Chain

Three anti-OPGL hybridoma light chain cDNAs (9H7, 16E1 and 18B2) werecloned into pDSR19 mammalian cell expression vector. The construction ofa plasmid encoding the 9H7 kappa light chain is explicitly described;cloning of the other light chain species was performed using similarprocedures. The anti-OPGL-9H7 kappa light chain variable region wasobtained using polymerase chain reaction (PCR) amplification methodsfrom first strand cDNA prepared from hybridoma 9H7 total RNA. Firststrand cDNA was prepared from 9H7 total RNA using a random primer withan extended 5′-adapter (5′-GGCCGGATAGGCCTCACT-3′, SEQ ID NO: 53) and thematerials and methods provided by the Gibco SuperScript II™Preamplification System for First Strand cDNA Synthesis kit (CatalogueNo. 18089-011). The oligonucleotides below were used for the PCR:

5′ GeneRacer ™ (Invitrogen) primer (SEQ ID NO: 54): 5′-GGA CAC TGA CAT GGA CTG AAG GAG TA- 3′; 3′ kappa RACE primer, 2310-03(SEQ ID NO: 55): 5′ -GGG GTC AGG CTG GAA CTG AGG- 3′.

The amplified DNAs were cloned into pCRII-TOPO (Invitrogen) and theresulting plasmids were sequenced. The kappa chain consensus sequencewas used to design primers for PCR amplification of the variable regionof the 9H7 kappa chain. To generate the signal sequence, a three-stepPCR was performed. First, primers 2669-73 and 2708-53 (set forth below)were used with a 9H7 cDNA light chain clone template. Conditions usedfor the reaction were: 94° C. for 1 minute; 94° C. for 20 seconds, 42°C. for 30 seconds, 74° C. for 150 seconds for 2 cycles; 94° C. for 20seconds, 56° C. for 30 seconds, 74° C. for 150 seconds for 25 cycles;and 74° C. for 7 minutes with Pfu polymerase and the appropriate bufferand nucleotides. The PCR product was then amplified with primers 2663-07and 2708-53 followed by amplification with primers 2663-08 and 2708-53.These primers are shown below.

2663-08 (SEQ ID NO: 56)             HindIII XbaI Kozak5′-C AGC AG AAGCTTCTAGA CCACC ATG GAC ATG AGG GTG CCC GCT CAG CTC CTG GG-3′; 2663-07 (SEQ ID NO: 57)5′-CC GCT CAG CTC CTG GGG CTC CTG CTG CTG TGG  CTG AGA GGT GCC AGA T-3′;2669-73 (SEQ ID NO: 58) 5′-G TGG TTG AGA GGT GCC AGA TGT GAA ATT GTG CTG ACC CAG TCT CCA GCC ACC CTG TCT TTG TCT  C-3′; 2708-53(SEQ ID NO: 59)          SalI5′-CTT GTC GAC TCA ACA CTC TCC CCT GTT GAA  GCT C-3′.

The PCR reactions generated a 741 bp fragment encoding 238 amino acidresidues (including the 22 amino acid signal sequence) that was purifiedusing a QIAquick PCR Purification kit (Qiagen Cat. No. 28104), cut withXbaI and SalI, and Qiagen purified again. This fragment, containing thecomplete light chain with a 5′ Kozak (translational initiation) site andthe following signal sequence for mammalian expression:

MDMRVPAQLLGLLLLWLRGARC, (SEQ ID NO: 60)was ligated into pDSRα19 to generate plasmid pDSRα19:9H7 kappa (FIG.17). pDSRα19 has been described previously (see InternationalApplication, Publication No. WO 90/14363, which is herein incorporatedby reference for any purpose). Briefly, to make pDSRα19, pDSRα2 wasmodified in the following ways: the sequence containing thetranscription termination/polyadenylation signal from the alpha subunitof the bovine pituitary glycoprotein hormone alpha-FSH(follicle-stimulating hormone) was shortened by approximately 1400 basepairs, to 885 base pairs, and ends at the NdeI site after modification;the dihydrofolate reductase (DHFR) promoter contained 209 base pairs,having been shortened from the 5′ end by approximately 1 kilobase; andan approximately 550 base pair Bg/II fragment from the DHFR polyAsequence was deleted.

The 9H7 kappa light chain expression clone was sequenced to confirm thatit encoded the same peptide that was identified in the 9H7 hybridoma.The final expression vector, pDSRα19:9H7 kappa is 5479 bp and containsthe 7 functional regions described in Table 2.

TABLE 2 Plasmid Base Pair Number: 2 to 881 A transcriptiontermination/polyadenylation signal from the α-subunit of the bovinepituitary glycoprotein hormone (α-FSH) (Goodwin, et al., 1983, NucleicAcids Res. 11: 6873-82; Genbank Accession Number X00004) 882 to 2027 Amouse dihydrofolate reductase (DHFR) minigene containing the endogenousmouse DHFR promoter, the cDNA coding sequences, and the DHFRtranscription termination/polyadenylation signals (Gasser et al, 1982,Proc. Natl. Acad. Sci. U.S.A. 79: 6522-6; Nunberg et al., 1980, Cell 19:355-64; Setzer et al., 1982, J. Biol. Chem. 257: 5143-7; McGrogan etal., 1985, J. Biol. Chem. 260: 2307-14) 2031 to 3947 pBR322 sequencescontaining the ampicillin resistance marker gene and the origin forreplication of the plasmid in E. coli (Genbank Accession Number J01749)3949 to 4292 An SV40 early promoter, enhancer and origin of replication(Takebe et al., 1988, Mol. Cell Biol. 8: 466-72, Genbank AccessionNumber J02400) 4299 to 4565 A translational enhancer element from theHTLV-1 LTR domain (Seiki et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:3618-22, Genbank Accession Number J02029) 4574 to 4730 An intron fromthe SV40 16S, 19S splice donor/acceptor signals (Okayama and Berg, 1983.Mol. Cell Biol. 3: 280-9, Genbank Accession Number J02400) 4755 to 5479The 9H7D4 kappa light chain cDNA between the Xba1 and Sal1 sitesConstruction of pDSR19:hIgG1 C_(H)

A pDSR19: rat variable region/human constant region IgG₁ plasmid wasconstructed using a three-piece ligation of a rat variable regionsequence, the human constant region (CH1, hinge, CH2, and CH3 domains)and pDSR19. The linear pDSRα19:hIgG1 C_(H) plasmid was prepared bydigesting the pDSR19:rat variable region/human constant region IgG₁plasmid with restriction enzymes XbaI and BsmBI to remove the codingportion of the rat variable region. The resulting linear plasmidcontaining the 1.0 kbp human IgG₁ constant region domain (C_(H)1, hinge,C_(H)2 and C_(H)3 domains) was gel isolated and used to accept hybridomaderived αOPGL variable regions.

Cloning of the 9H7 Anti-OPGL MAb Heavy Chain

Three anti-OPGL hybridoma IgG₁ heavy chain cDNAs; 9H7, 16E1 and 18B2,were cloned into pDSR19 mammalian cell expression vector. Theconstruction of a plasmid encoding the 9H7 IgG₁ heavy chain isexplicitly described here; the other hybridoma heavy chains were clonedusing similar procedures. The anti-OPGL-9H7 heavy chain variable regionwas obtained using PCR amplification methods from first strand cDNAprepared from hybridoma 9H7 total RNA. First strand cDNA was preparedfrom 9H7 total RNA using a random primer with an extended 5′-adapter(5′-GGCCGGATAGGCCTCACT-3′, SEQ ID NO: 53) and the materials and methodsprovided by the Gibco SuperScript II™ Preamplification System for FirstStrand cDNA Synthesis kit (Cat. No. 18089-011). The oligonucleotidesbelow were used for the PCR:

5′ heavy chain RACE primer, 2508-02 (SEQ ID NO: 61): 5′-(CG)AG GT(CG) CAG (CT)T(GT) GTG (CG)AG TC- 3′; 3′heavy chain RACE primer, 2420-54 (SEQ ID NO: 62): 5′-CTG AGT TCC ACG ACA CC- 3′.

Amplified DNA was cloned into pCRII-TOPO (Invitrogen) and the resultingplasmids were sequenced. The heavy chain consensus sequence was used todesign primers for PCR amplification of the variable region of the 9H7heavy chain. To generate the signal sequence, a three-step PCR wasperformed. First, primers 2512-98 and 2673-14 were used with a 9H7 heavychain cDNA clone template. Conditions used for the reaction were: 94° C.for 1 minute; 94° C. for 20 seconds, 42° C. for 30 seconds, 74° C. for150 seconds for 2 cycles; 94° C. for 20 seconds, 56° C. for 30 seconds,74° C. for 150 seconds for 25 cycles; and 74° C. for 7 minutes with Pfupolymerase and the appropriate buffer and nucleotides. The PCR productwas then amplified with primers 2663-07 and 2673-14 followed byamplification with primers 2663-08 and 2673-14. The primers are shownbelow.

2663-08 (SEQ ID NO: 63)             HindIII XbaI Kozak5′-C AGC AG AAGCTTCTAGA CCACC ATG GAC ATG AGGGTG CCC GCT CAG CTC CTG GG-3′; 2663-07 (SEQ ID NO: 64)5′-CC GCT CAG CTC CTG GGG CTC CTG CTG CTG TGG CTG AGA GGT GCC AGA T-3′;2512-98 (SEQ ID NO: 65) 5′-G TGG TTG AGA GGT GCC AGA TGT GAG GTG CAG CTG GTG CAG TCT -3′; 2673-14 (SEQ ID NO: 66)                     BsmBI5′-GT GGA GGC ACT AGA GAC GGT GAC CAG GGC TCC CTG GCC CCA GGG GTC GAA -3′.

The PCR reactions generated a 443 bp fragment encoding 138 amino acidresidues (including the 22 amino acid signal sequence) that was purifiedusing a QIAquick PCR Purification kit (Qiagen Cat. No. 28104), cut withXbaI and BsmBI, and Qiagen purified again. This fragment, containing theheavy chain with a 5′ Kozak (translational initiation) site and thefollowing signal sequence for mammalian expression:

MDMRVPAQLLGLLLLWLRGARC, (SEQ ID NO: 60)was ligated into pDSRα19:hIgG1 C_(H) to generate plasmid pDSRα19:9H7IgG1 (FIG. 18).

The 9H7 IgG₁ heavy chain expression clone was sequenced to confirm thatit encoded the same peptide that was identified in the 9H7 hybridoma.The final expression vector, pDSRα19:rat variable region/human constantregion IgG₁ is 6158 bp and contains the 7 functional regions describedin Table 3.

TABLE 3 Plasmid Base Pair Number:    2 to 881A transcription termination/polyadenylation signal from the α-subunit of the bovinepituitary glycoprotein hormone (α-FSH) (Goodwin, et al., 1983, Nucleic Acids Res.11: 6873-82; Genbank Accession Number X00004)  882 to 2027A mouse dihydrofolate reductase (DHFR) minigene containing the endogenousmouse DHFR promoter, the cDNA coding sequences, and the DHFR transcriptiontermination/polyadenylation signals (Gasser et al, 1982, Proc. Natl. Acad. Sci.U.S.A. 79: 6522-6; Nunberg et al., 1980, Cell 19: 355-64; Setzer et al., 1982, J. Biol. Chem. 257: 5143-7; McGrogan et al., 1985, J. Biol. Chem. 260: 2307-14)2031 to 3947pBR322 sequences containing the ampicillin resistance marker gene and the originfor replication of the plasmid in E. coli (Genbank Accession Number J01749)3949 to 4292An SV40 early promoter, enhancer and origin of replication (Takebe et al., 1988,Mol. Cell Biol. 8: 466-72, Genbank Accession Number J02400) 4299 to 4565A translational enhancer element from the HTLV-1 LTR domain(Seiki et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80: 3618-22, Genbank AccessionNumber J02029) 4574 to 4730An intron from the SV40 16S, 19S splice donor/acceptor signals (Okayama andBerg, 1983. Mol. Cell Biol. 3: 280-9, Genbank Accession Number J02400)4755 to 6158The rVh/hCh1 heavy chain cDNA between the XbaI and SalI sites. The sequencesof which follows (SEQ ID NO: 67): XbaITCTAG ACCACCATGG ACATCAGGCT CAGCTTAGTT TTCCTTGTCCTTTTCATAAA AGGTGTCCAG TGTGAGGTAG AACTGGTGGAGTCTGGGGGC GGCTTAGTAC AACCTGGAAG GTCCATGACACTCTCCTGTG CAGCCTCGGG ATTCACTTTC AGAACCTATGGCATGGCCTG GGTCCGCCAG GCCCCAACGA AGGGTCTGGAGTGGGTCTCA TCAATTACTG CTAGTGGTGG TACCACCTACTATCGAGACT CCGTGAAGGG CCGCTTCACT ATTTTTAGGGATAATGCAAA AAGTACCCTA TACCTGCAGA TGGACAGTCCGAGGTCTGAG GACACGGCCA CTTATTTCTG TACATCAATT                                   BsmBITCGGAATACT GGGGCCACGG AGTCATGGTC ACCGTCTCTAGTGCCTCCACCAAGGGCCCA TCGGTCTTCC CCCTGGCACCCTCCTCCAAG AGCACCTCTGGGGGCACAGC GGCCCTGGGCTGCCTGGTCA AGGACTACTT CCCCGAACCG GTGACGGTGTCGTGGAACTC AGGCGCCCTG ACCAGCGGCG TGCACACCTTCCCGGCTGTC CTACAGTCCT CAGGACTCTA CTCCCTCAGCAGCGTGGTGACCGTGCCCTC CAGCAGCTTG GGCACCCAGACCTACATCTG CAACGTGAATCACAAGCCCA GCAACACCAAGGTGGACAAG AAAGTTGAGC CCAAATCTTG TGACAAAACTCACACATGCC CACCGTGCCC AGCACCTGAA CTCCTGGGGGGACCGTCAGT CTTCCTCTTC CCCCCAAAAC CCAAGGACACCCTCATGATC TCCCGGACCC CTGAGGTCAC ATGCGTGGTGGTGGACGTGA GCCACGAAGACCCTGAGGTC AAGTTCAACTGGTACGTGGA CGGCGTGGAG GTGCATAATG CCAAGACAAAGCCGCGGGAG GAGCAGTACA ACAGCACGTA CCGTGTGGTCAGCGTCCTCA CCGTCCTGCA CCAGGACTGG CTGAATGGCAAGGAGTACAAGTGCAAGGTC TCCAACAAAG CCCTCCCAGCCCCCATCGAG AAAACCATCTCCAAAGCCAA AGGGCAGCCCCGAGAACCAC AGGTGTACAC CCTGCCCCCA TCCCGGGATGAGCTGACCAA GAACCAGGTC AGCCTGACCT GCCTGGTCAAAGGCTTCTAT CCCAGCGACA TCGCCGTGGA GTGGGAGAGCAATGGGCAGCCGGAGAACAA CTACAAGACC ACGCCTCCCGTGCTGGACTC CGACGGCTCC TTCTTCCTCT ATAGCAAGCTCACCGTGGAC AAGAGCAGGT GGCAGCAGGG GAACGTCTTCTCATGCTCCG TGATGCATGA GGCTCTGCAC AACCACTACACGCAGAAGAG CCTCTCCCTG TCTCCGGGTA          SalI AATGATAAGT CGAC

Example 3 9H7 Anti-OPGL MAb Expression in CHO Cells

Recombinant anti-OPGL antibodies are produced by Chinese hamster ovarycells, specifically CHO AM-1/D, as disclosed in U.S. Pat. No. 6,210,924(incorporated by reference). DNA sequences encoding the complete heavyor light chains of each anti-OPGL antibody of the invention are clonedinto expression vectors such as those described above. CHO AM-1/D cellsare co-transfected with an expression vector capable of expressing acomplete heavy chain and an expression vector expressing the completelight chain of the appropriate anti-OPGL antibody. For example, togenerate the 22B3 antibody, cells are co-transfected with a vectorcapable of expressing a complete heavy chain comprising the amino acidsequence as set forth in SEQ ID NO: 30 and a vector capable ofexpressing a complete light chain comprising the amino acid sequence setforth in SEQ ID NO: 32. To generate the 2E11 antibody, cells areco-transfected with a vector capable of expressing a complete heavychain comprising the amino acid sequence as set forth in SEQ ID NO: 34and a vector capable of expressing a complete light chain comprising theamino acid sequence set forth in SEQ ID NO: 36. To generate the 2D8antibody, cells are co-transfected with a vector capable of expressing acomplete heavy chain comprising the amino acid sequence as set forth inSEQ ID NO: 38 and a vector capable of expressing a complete light chaincomprising the amino acid sequence set forth in SEQ ID NO: 40. Togenerate the 18B2 antibody, cells are co-transfected with a vectorcapable of expressing a complete heavy chain comprising the amino acidsequence as set forth in SEQ ID NO: 42 and a vector capable ofexpressing a complete light chain comprising the amino acid sequence setforth in SEQ ID NO: 44. To generate the 16E1 antibody, cells areco-transfected with a vector capable of expressing a complete heavychain comprising the amino acid sequence as set forth in SEQ ID NO: 46and a vector capable of expressing a complete light chain comprising theamino acid sequence set forth in SEQ ID NO: 48. To generate the 9H7antibody, cells are co-transfected with a vector capable of expressing acomplete heavy chain comprising the amino acid sequence as set forth inSEQ ID NO: 50 and a vector capable of expressing a complete light chaincomprising the amino acid sequence set forth in SEQ ID NO: 52. Table 4summarizes the complete heavy and complete light chains for the variousOPGL antibodies.

TABLE 4 Heavy Chain Variable Region + Antibody Heavy Chain ConstantRegion Complete Heavy Chain 22B3 SEQ ID NO: 6 + SEQ ID NO: 2 SEQ ID NO:30 2E11 SEQ ID NO: 10 + SEQ ID NO: 2 SEQ ID NO: 34 2D8 SEQ ID NO: 14 +SEQ ID NO: 2 SEQ ID NO: 38 18B2 SEQ ID NO: 18 + SEQ ID NO: 2 SEQ ID NO:42 16E1 SEQ ID NO: 22 + SEQ ID NO: 2 SEQ ID NO: 46 9H7 SEQ ID NO: 26 +SEQ ID NO: 2 SEQ ID NO: 50 Light Chain Variable Region + Antibody LightChain Constant Region Complete Light Chain 22B3 SEQ ID NO: 8 + SEQ IDNO: 4 SEQ ID NO: 32 2E11 SEQ ID NO: 12 + SEQ ID NO: 4 SEQ ID NO: 36 2D8SEQ ID NO: 16 + SEQ ID NO: 4 SEQ ID NO: 40 18B2 SEQ ID NO: 20 + SEQ IDNO: 4 SEQ ID NO: 44 16E1 SEQ ID NO: 24 + SEQ ID NO: 4 SEQ ID NO: 48 9H7SEQ ID NO: 28 + SEQ ID NO: 4 SEQ ID NO: 52

Stable expression of the 9H7 anti-OPGL MAb was achieved byco-transfection of pDSRα19:9H7 IgG₁ and pDSRα19:9H7 kappa plasmids intodihydrofolate reductase deficient (DHFR⁻) serum-free adapted Chinesehamster ovary cells (CHO AM-1/D, U.S. Pat. No. 6,210,924) using theart-recognized calcium phosphate method. Transfected cells were selectedin 96 well plates in medium containing dialyzed serum but not containinghypoxanthine-thymidine to ensure the growth of cells expressing the DHFRenzyme. Over 5000 transfected clones were screened using assays such asHTRF (homogeneous time resolved fluorescence) and ELISA in order todetect expression of 9H7 anti-OPGL MAb in the conditioned medium. Thehighest expressing clones were selected for single cell cloning andcreation of cell banks.

Example 4 Production of Anti-OPGL Antibodies

Anti-OPGL antibodies are produced by expression in a clonal line of CHOcells. For each production run, cells from a single vial are thawed intoserum-free cell culture media. The cells are grown initially in aT-flask and are serially expanded through a series of spinner flasksuntil sufficient inoculum has been generated to seed a 20 L bioreactor.Following growth for 5-10 days, the culture is then used to inoculate a300 L bioreactor. Following growth for an additional 5-10 days, theculture is used to inoculate a 2000 L bioreactor. Production is carriedout in a 2000 L bioreactor using a fed batch culture, in which anutrient feed containing concentrated media components is added tomaintain cell growth and culture viability. Production lasts forapproximately two weeks during which time anti-OPGL antibody isconstitutively produced by the cells and secreted into the cell culturemedium.

The production reactor is controlled at set pH, temperature, anddissolved oxygen level: pH is controlled by carbon dioxide gas andsodium carbonate addition; dissolved oxygen is controlled by air,nitrogen, and oxygen gas flows.

At the end of production, the cell broth is fed into a disk stackcentrifuge and the culture supernatant is separated from the cells. Theconcentrate is further clarified through a depth filter followed by a0.2 μm filter. The clarified conditioned media is then concentrated bytangential flow ultrafiltration. The conditioned media is concentrated15- to 30-fold. The resulting concentrated conditioned medium is theneither processed through purification or frozen for purification at alater date. FIG. 19 depicts an exemplary cell culture process forproducing an anti-OPGL antibody.

Example 5 Screening of Antibodies for Binding to OPGL by BIAcore

All experiments were performed on a BIAcore 2000 according to themanufacturer's instructions, with the following modifications.Experiments were performed at room temperature using a running buffercontaining 10 mM Hepes (pH 7.4), 0.15M NaCl, 3 mM EDTA, and 0.005% Tween20. Protein G at 50 μg/mL in 10 mM acetate pH 4.5 was immobilized to alevel of 1,600 response units (RU) onto a CM5 Research grade sensor chip(BIAcore, Inc.). Antibodies (8-20 μg/mL) were captured onto the ProteinG chip at a level of 300-400 RUs. CHO human OPGL (hOPGL) 140 or E. colimouse OPGL (mOPGL) 158 were passed over the immobilized antibodies atconcentrations of 0.25-62 nM. Langmuir 1:1 model was used to determinebinding kinetics. Protein G immobilized to 1600 RUs was used as a blanksurface. A mouse monoclonal antibody was used as a positive control toshow binding to hOPGL 140 and to monitor surface stability.

All anti-OPGL antibodies showed strong binding to CHO hOPGL 140. 22B3appears to have a slower off rate than the other antibodies tested. Nobinding of E. coli mOPGL 158 was detected. The results are summarized inTable 5.

TABLE 5 hOPGL 140 off rate half life mOPGL Ab ka (1/Ms) kd (1/s) KD(1/M) KD t½ (s) 158 9H7 1.27E+06 2.26E−04 1.93E−10 190 pm 3067 nobinding 18B2 8.78E+05 1.86E−04 2.11E−10 210 pm 3726 no binding 2D81.97E+06 1.81E−04 9.20E−11  92 pm 3829 no binding 2E11 4.53E+05 1.32E−042.92E−10 290 pm 5251 no binding 16E1 2.16E+06 1.37E−04 6.33E−11  63 pm5059 no binding 22B3 1.90E+06 6.39E−05 3.37E−11  34 pm 10847 no binding

Example 6 Anti-OPGL Antibody Neutralizing Activity Inhibition ofOsteoclast Formation

RAW 264.7 (Accession No. TIB-71, American Type Culture Collection,Manassas, Va.) is a murine macrophage cell line that was derived from anAbelson murine leukemia virus-induced tumor. RAW 264.7 cells willdifferentiate to osteoclast-like cells in the presence of OPGL. An assayfor generation of osteoclasts in culture from RAW cells in the presenceof OPGL has been described in detail by Hsu et al., 1999, Proc. Natl.Acad. Sci. U.S.A. 96:3540-3545, which is incorporated by referenceherein.

RAW cells can be stimulated by OPGL ligand to differentiate intoosteoclast-like cells, and the differentiation can be measured bytartrate-resistant acid phosphatase (TRAP) activity, a property ofosteoclasts. This activity provides the basis for characterizinganti-OPGL antibodies produced according to the invention, by assayingthe effect of said antibodies on osteoclastogenesis.

RAW cells were incubated for 4 days in the presence of a constant amountof OPGL (40 ng/mL) and varying amounts of anti-OPGL antibody (6.3 ng/mLto 200 ng/mL) in cell culture medium (DMEM, 10% FBS, 0.3 mg/mLL-glutamine, 100 units/mL penicillin G, 100 μg/mL streptomycin sulfate).At the end of 4 days, the cells were stained for tartrate-resistant acidphosphatase (TRAP) activity by permeabilization and acidification,followed by treatment with p-nitrophenylphosphate (PNPP) for 5 minutes.Briefly, the media was aspirated from the cells, and 100 μL of citratebuffer (having a formula of 410 mL 0.1M citric acid, 590 mL 0.1 Mcitrate, trisodium salt and 1 mL Triton X-100) was added to each welland the plates incubated for 3 to 5 minutes at room temperature. Onehundred microliters of PNPP solution (having a formula of 157.8 mg acidphosphatase reagent (Sigma 104-100), 7.2 mL tartrate solution (SigmaCat. No. 387-3), and 22.8 mL citrate buffer) was then added, and plateswere incubated for 3 to 5 minutes at room temperature. The reaction wasterminated by addition of 50 μL 0.5 M NaOH solution.

TRAP converts p-nitrophenylphosphate to para-nitrophenol, which can bequantitated by optical density measurement at 405 nm. The TRAP activity,which is a surrogate marker for osteoclast development, thereforecorrelates with the optical density at 405 nm. A plot of optical densityversus anti-OPGL antibody concentration is shown in FIG. 20, anddemonstrates that anti-OPGL antibody inhibited osteoclast formation inthis assay in a dose-dependent manner. IC₅₀ values were calculated usingthe forecast function, and are shown in Table 6. An alkalinephosphatase-linked rat polyclonal anti-human OPGL antibody (APRa-anti-HuOPGL Ab) with OPGL neutralizing activity was used as apositive control for the anti-huOPGL antibody neutralizing activityassay.

TABLE 6 Sample IC₅₀ (ng/mL) AP Ra-anti-HuOPGL Ab 112 9H7 129 18B2 80 2D8611 2E11 77 16E1 352 22B3 146 IC₅₀ (ng/mL) Running Average APRa-anti-HuOPGL Ab Average 140 Stdev 35.8 CV 26% Count 25

Example 7 Pharmacokinetics in Cynomolgus Monkeys

The in vivo activity and pharmacokinetics of the anti-OPGL antibodies ofthe invention were assayed using cynomolgus monkeys. Three femalecynomolgus monkeys, not greater than 5 years of age and weighing 2 to 5kg each received single subcutaneous (SC) doses of 1 mg/kg anti-OPGLantibody.

Animals were dosed with anti-OPGL antibody expressed from transfectedChinese hamster ovary (CHO) cells and serum samples were taken fordetermination of anti-OPGL antibody levels, anti-therapeutic antibodyanalysis, and analysis of the bone turnover marker serum N-telopeptide(serum N-Tx), alkaline phosphatase (ALP), and serum calcium (serum Ca).

The serum concentration-time profiles following SC administration areshown in FIG. 21. The serum N-Tx concentration-time profiles followingSC administration are shown in FIG. 22.

Example 8 Identification of an Epitope for Antibodies on OPGL Productionof Variant Murine OPGL

Human OPGL [143-317] was produced as described in Example 1 of WO01/62932, published Aug. 30, 2001, which is hereby incorporated byreference in its entirety. Murine OPGL [158-316] containing amino acidresidues 158 through 316 of murine OPGL (as shown in FIG. 1 ofInternational Application, Publication No. W098/46751, incorporated byreference) preceded by an introduced N-terminal methionine residue wasproduced in E. coli. Murine OPGL [158-316] was purified from the solublefraction of bacteria as described previously (Lacey et al., 1998, Cell93:165-176). FLAG-tagged murine OPGL [158-316] was produced byintroducing a nucleic acid encoding an N-terminal methionine followed bya FLAG-tag sequence fused to the N-terminus of residues 158-316 as shownin FIG. 1 of International Application, Publication No. WO98/46751 usingconventional genetic engineering techniques. The FLAG-tagged OPGL[158-316] molecule was cloned into bacterial expression vector pAMG21(pAMG21 was deposited with the American Type Culture Collection andhaving Accession No. 98113).

A FLAG-tagged murine OPGL [158-316] polypeptide variant was constructedin which amino acid residues SVPTD (SEQ ID NO: 68) at positions 229-233(as shown in FIG. 1 of International Application, Publication No.WO98/46751) were substituted with corresponding amino acid residuesDLATE (SEQ ID NO: 69) at positions 230-234 (as shown in FIG. 4 ofInternational Application, Publication No. WO98/46751). The resultingconstruct referred to as “FLAG-murine OPGL [158-316]/DE” has the nucleicacid and protein sequence as shown in FIG. 23 (SEQ ID NO: 72) (whichshows only where the mutations are located). The amino acid sequencechanges are located in a region of OPGL between the D and E regions.FIG. 23 shows a comparison of murine (SEQ ID NO: 70), human (SEQ ID NO:71), and murine DE variant (SEQ ID NO: 72) amino acid sequences in thisregion. The sequence changes in the murine variant are S231D, V232L,P233A and D235E with the T at position 234 unchanged. Flanking sequencesin this region are virtually identical between murine and human OPGL.

This molecule was constructed using a two-step PCR reaction where thefirst step contained two separate PCR reactions, designated reaction Aand reaction B. For both reaction A and reaction B, pAMG21-FLAG-murineOPGL [158-316] DNA was used as a template for PCR. Reaction A employedoligonucleotides #2640-90 and #2640-91 for PCR, whereas reaction Bemployed oligonucleotides #2640-92 and #2640-93.

#2640-90 (SEQ ID NO: 73): CCTCTCATATGGACTACAAGGAC;#2640-91 (SEQ ID NO 74): AGTAGCCAGGTCTCCCGATGTTTCATGATG;#2640-92 (SEQ ID NO: 75): CTGGCTACTGAATATCTTCAGCTGATGGTG;#2640-93 (SEQ ID NO: 76): CCTCTCCTCGAGTTAGTCTATGTCC.

Conditions for reactions A and B were: 95° C. for 1 min; 95° C. for 20seconds, 44° C. for 30 seconds, 72° C. for 45 seconds for 5 cycles; 95°C. for 20 seconds, 60° C. for 30 seconds, 72° C. for 45 seconds for 25cycles; and 72° C. for 10 minutes with Pfu Turbo polymerase (Stratagene)and the appropriate buffer and nucleotides. After thermocycling wasperformed, PCR products from reactions A and B were purified from anagarose gel using conventional methods. The second step PCR reaction,designated reaction C, utilized purified reaction A and reaction B PCRproducts as a template and oligonucleotides #2640-90 and #2640-93 asprimers. Conditions for reaction C were: 95° C. for 1 minute; 95° C. for20 seconds, 37° C. for 30 seconds, 72° C. for 1 minute for 25 cycles;and 72° C. for 10 minutes with Pfu Turbo polymerase and the appropriatebuffer and nucleotides. Following thermocycling, the product fromreaction C was cloned into the pCRII-TOPO cloning vector (Invitrogen)and electroporated into DH10b (Gibco) cells using methods provided bythe manufacturer. Clones were selected and sequenced to confirm theamino acid sequence SVPTD (SEQ ID NO: 68) in murine OPGL [158-316] waschanged to DLATE (SEQ ID NO: 69). The sequence-verified DNA was thendigested with NdeI and XhoI, purified, and subcloned into bacterialexpression vector pAMG21 giving rise to plasmid pAMG21-FLAG-murineOPGL[158-316]/DE.

E. coli host GM94 (deposited with the American Type Culture Collectionunder Accession No. 202173) containing plasmid pAMG21-FLAG-murineOPGL[158-316]/DE was grown in 2XYT media to an exponential growth phaseand induced to express the FLAG-tagged murine OPGL[158-316]/DE proteinby addition of V. fischeri synthetic autoinducer to 100 ng/mL.Approximately 3-6 hours after induction, the cells were pelleted andrecombinant FLAG-murine OPGL[158-316]/DE protein was purified from thesoluble fraction of E. coli using methods described in Lacey et al.,ibid.

Binding of Anti-Human OPGL Antibodies to Human OPGL[143-317], MurineOPGL[158-316], and FLAG-Murine OPGL[158-316]/DE

Costar E.I.A./R.I.A. Plates (Flat Bottom High Binding, Cat #3590) werecoated with 100 μL/well of either human OPGL[143-317] protein, murineOPGL[158-316] protein, or FLAG-tagged murine OPGL[158-316]/DE protein at3 μg/mL in PBS, overnight at 4° C. with agitation. After overnightincubation, the protein solutions were removed from the plate and 200 μLof 5% Chicken Serum (Gibco/BRL Cat #16110-082) in PBST (PBS plus 0.05%Tween 20) was added to each well of the plate and plates were incubatedat room temperature (RT) for 3 hours with agitation. After incubationand blocking, plates were washed 4 times with 1× K-P wash solution indH₂O (Cat #50-63-00, Kirkegaard & Perry Laboratories) and dried.Purified anti-OPGL antibody or human OPGL [22-194]-Fc protein wasserially diluted 1:1 from 2 μg/mL to 1.953 ng/mL in 5% Chicken Serum inPBST and 100 μL/well was added to appropriate wells of the microtiterplate coated with either human OPGL[143-317], murine OPGL[158-316], orFLAG-tagged murine OPGL[158-316]/DE protein. Plates were incubated for2.25 hours at room temperature with agitation, washed four times with 1×K-P wash solution and dried. Goat anti-human IgG (Fc) (JacksonImmunoResearch, Cat #109-036-098) was diluted 1:3000 in 5% Chicken Serumin PBST and 100 μL was added to each well. Plates were incubated for1.25 hours at room temperature with agitation, washed six times with 1X× K-P wash solution, and dried. 100 μL of undiluted ABTS substrate(Kirkegaard & Perry; Cat #50-66-00) was added to each well and the dishwas incubated at room temperature until sufficient blue-green colordeveloped. Color development was stopped by addition of 100 μL 1% SDS.Quantitation of color development was performed using a microtiter platereader with detection at 405 nm.

The results of the enzyme immunoassay are shown in FIGS. 24 and 25. Allsix anti-OPGL antibodies of the invention bind to human OPGL[143-317].However, only 22B3 antibody shows detectable binding to murineOPGL[158-316] over the concentration range tested (FIG. 24). Whilebinding of 22B3 antibody to murine OPGL[158-316] occurs with a muchlower affinity than to human OPGL[143-317], the 2D8, 9H7, 16E1, and 22B3antibodies bind to FLAG-tagged murine OPGL[158-316]/DE (FIG. 25) almostas well as to human OPGL[143-317] under the assay conditions above.Thus, the amino acid changes in murine OPGL[158-316]/DE compared tomurine OPGL[158-316] are important to the binding activity of antibodies2D8, 9H7, 16E1, and 22B3. Antibodies 2E11 and 18B2 show no detectablebinding to either murine OPGL[158-316] or murine OPGL[158-316]/DE.

The FLAG-murine OPGL[158-316]/DE was assayed for activity in a RAW cellassay as described in Example 6 and observed to have a similar ED50 forosteoclast formation as human OPGL[143-317], indicating that the DEvariant is active in promoting osteoclast activity in vitro. Therefore,the binding of the anti-OPGL antibodies to murine OPGL[158-316]/DE islikely to inhibit osteoclast formation.

The epitope of the 2D8, 9H7, 16E1, and 22B3 anti-human OPGL antibodiesis located to a region of human OPGL that includes at least amino acidresidues DLATE (residues 230 through 234 of human OPGL as shown in FIG.4 of International Application, Publication No. W098/46751) termed theD-E loop. The 2E11 and 18B2 anti-human OPGL antibodies do not bind topeptide fragments corresponding to the D-E loop region by itself.However, it will be recognized that in the native molecule theseantibodies may bind to an epitope outside the D-E loop region or theymay bind to all or a portion of the D-E loop region in combination withother portions of the molecule.

It should be understood that the foregoing disclosure emphasizes certainspecific embodiments of the invention and that all modifications oralternatives equivalent thereto are within the spirit and scope of theinvention as set forth in the appended claims.

1. A method of treating an osteopenic disorder in a patient, comprisingadministering to a patient a pharmaceutically effective amount of anisolated human antibody that specifically binds osteoprotegerin ligand(OPGL), wherein the antibody comprises: (a) a heavy chain having a heavychain variable region comprising an amino acid sequence as set forth inSEQ ID NO: 6, an antigen-binding fragment thereof, or an immunologicallyfunctional immunoglobulin fragment thereof, and a light chain having alight chain variable region comprising an amino acid sequence as setforth in SEQ ID NO: 8, an antigen-binding fragment thereof, or animmunologically functional immunoglobulin fragment thereof; (b) a heavychain having a heavy chain variable region comprising an amino acidsequence as set forth in SEQ ID NO: 14, an antigen-binding fragmentthereof, or an immunologically functional immunoglobulin fragmentthereof, and a light chain having a light chain variable regioncomprising an amino acid sequence as set forth in SEQ ID NO: 16, anantigen-binding fragment thereof, or an immunologically functionalimmunoglobulin fragment thereof; (c) a heavy chain having a heavy chainvariable region comprising an amino acid sequence as set forth in SEQ IDNO: 22, an antigen-binding fragment thereof, or an immunologicallyfunctional immunoglobulin fragment thereof, and a light chain having alight chain variable region comprising an amino acid sequence as setforth in SEQ ID NO: 24, an antigen-binding fragment thereof, or animmunologically functional immunoglobulin fragment thereof; (d) a heavychain having a heavy chain variable region comprising an amino acidsequence as set forth in SEQ ID NO: 26, an antigen-binding fragmentthereof, or an immunologically functional immunoglobulin fragmentthereof, and a light chain having a light chain variable regioncomprising an amino acid sequence as set forth in SEQ ID NO: 28, anantigen-binding fragment thereof, or an immunologically functionalimmunoglobulin fragment thereof; (e) a heavy chain comprising an aminoacid sequence as set forth in SEQ ID NO: 30, an antigen-binding fragmentthereof, or an immunologically functional immunoglobulin fragmentthereof, and a light chain comprising an amino acid sequence as setforth in SEQ ID NO: 32, an antigen-binding fragment thereof, or animmunologically functional immunoglobulin fragment thereof; (f) a heavychain comprising an amino acid sequence as set forth in SEQ ID NO: 38,an antigen-binding fragment thereof, or an immunologically functionalimmunoglobulin fragment thereof, and a light chain comprising an aminoacid sequence as set forth in SEQ ID NO: 40, an antigen-binding fragmentthereof, or an immunologically functional immunoglobulin fragmentthereof; (g) a heavy chain comprising an amino acid sequence as setforth in SEQ ID NO: 46, an antigen-binding fragment thereof, or animmunologically functional immunoglobulin fragment thereof, and a lightchain comprising an amino acid sequence as set forth in SEQ ID NO: 48,an antigen-binding fragment thereof, or an immunologically functionalimmunoglobulin fragment thereof; or (h) a heavy chain comprising anamino acid sequence as set forth in SEQ ID NO: 50, an antigen-bindingfragment thereof, or an immunologically functional immunoglobulinfragment thereof, and a light chain comprising an amino acid sequence asset forth in SEQ ID NO: 52, an antigen-binding fragment thereof, or animmunologically functional immunoglobulin fragment thereof; wherein theantibody binding inhibits binding of OPGL to an osteoclastdifferentiation and activation receptor (ODAR).
 2. A method of treatingan osteopenic disorder in a patient, comprising administering to apatient a pharmaceutically effective amount of an isolated humanantibody that specifically binds osteoprotegerin ligand (OPGL), whereinthe antibody comprises: (a) a heavy chain having a heavy chain variableregion comprising an amino acid sequence as set forth in SEQ ID NO: 10,an antigen-binding fragment thereof, or an immunologically functionalimmunoglobulin fragment thereof, and a light chain having a light chainvariable region comprising an amino acid sequence as set forth in SEQ IDNO: 12, an antigen-binding fragment thereof, or an immunologicallyfunctional immunoglobulin fragment thereof; (b) a heavy chain having aheavy chain variable region comprising an amino acid sequence as setforth in SEQ ID NO: 18, an antigen-binding fragment thereof, or animmunologically functional immunoglobulin fragment thereof, and a lightchain having a light chain variable region comprising an amino acidsequence as set forth in SEQ ID NO: 20, an antigen-binding fragmentthereof, or an immunologically functional immunoglobulin fragmentthereof; (c) a heavy chain comprising an amino acid sequence as setforth in SEQ ID NO: 34, an antigen-binding fragment thereof, or animmunologically functional immunoglobulin fragment thereof, and a lightchain comprising an amino acid sequence as set forth in SEQ ID NO: 36,an antigen-binding fragment thereof, or an immunologically functionalimmunoglobulin fragment thereof; or (d) a heavy chain comprising anamino acid sequence as set forth in SEQ ID NO: 42, an antigen-bindingfragment thereof, or an immunologically functional immunoglobulinfragment thereof, and a light chain comprising an amino acid sequence asset forth in SEQ ID NO: 44, an antigen-binding fragment thereof, or animmunologically functional immunoglobulin fragment thereof; wherein theantibody binding inhibits binding of OPGL to an osteoclastdifferentiation and activation receptor (ODAR).
 3. A method of treatingan osteopenic disorder in a patient, comprising administering to apatient a pharmaceutical composition, wherein the pharmaceuticalcomposition comprises a pharmaceutically acceptable carrier and atherapeutically effective amount of an isolated human antibody thatspecifically binds osteoprotegerin ligand (OPGL), wherein the antibodycomprises: (a) a heavy chain having a heavy chain variable regioncomprising an amino acid sequence as set forth in SEQ ID NO: 6, anantigen-binding fragment thereof, or an immunologically functionalimmunoglobulin fragment thereof, and a light chain having a light chainvariable region comprising an amino acid sequence as set forth in SEQ IDNO: 8, an antigen-binding fragment thereof, or an immunologicallyfunctional immunoglobulin fragment thereof; (b) a heavy chain having aheavy chain variable region comprising an amino acid sequence as setforth in SEQ ID NO: 14, an antigen-binding fragment thereof, or animmunologically functional immunoglobulin fragment thereof, and a lightchain having a light chain variable region comprising an amino acidsequence as set forth in SEQ ID NO: 16, an antigen-binding fragmentthereof, or an immunologically functional immunoglobulin fragmentthereof; (c) a heavy chain having a heavy chain variable regioncomprising an amino acid sequence as set forth in SEQ ID NO: 22, anantigen-binding fragment thereof, or an immunologically functionalimmunoglobulin fragment thereof, and a light chain having a light chainvariable region comprising an amino acid sequence as set forth in SEQ IDNO: 24, an antigen-binding fragment thereof, or an immunologicallyfunctional immunoglobulin fragment thereof; (d) a heavy chain having aheavy chain variable region comprising an amino acid sequence as setforth in SEQ ID NO: 26, an antigen-binding fragment thereof, or animmunologically functional immunoglobulin fragment thereof, and a lightchain having a light chain variable region comprising an amino acidsequence as set forth in SEQ ID NO: 28, an antigen-binding fragmentthereof, or an immunologically functional immunoglobulin fragmentthereof; (e) a heavy chain comprising an amino acid sequence as setforth in SEQ ID NO: 30, an antigen-binding fragment thereof, or animmunologically functional immunoglobulin fragment thereof, and a lightchain comprising an amino acid sequence as set forth in SEQ ID NO: 32,an antigen-binding fragment thereof, or an immunologically functionalimmunoglobulin fragment thereof; (f) a heavy chain comprising an aminoacid sequence as set forth in SEQ ID NO: 38, an antigen-binding fragmentthereof, or an immunologically functional immunoglobulin fragmentthereof, and a light chain comprising an amino acid sequence as setforth in SEQ ID NO: 40, an antigen-binding fragment thereof, or animmunologically functional immunoglobulin fragment thereof; (g) a heavychain comprising an amino acid sequence as set forth in SEQ ID NO: 46,an antigen-binding fragment thereof, or an immunologically functionalimmunoglobulin fragment thereof, and a light chain comprising an aminoacid sequence as set forth in SEQ ID NO: 48, an antigen-binding fragmentthereof, or an immunologically functional immunoglobulin fragmentthereof; or (h) a heavy chain comprising an amino acid sequence as setforth in SEQ ID NO: 50, an antigen-binding fragment thereof, or animmunologically functional immunoglobulin fragment thereof, and a lightchain comprising an amino acid sequence as set forth in SEQ ID NO: 52,an antigen-binding fragment thereof, or an immunologically functionalimmunoglobulin fragment thereof; wherein the antibody binding inhibitsbinding of OPGL to an osteoclast differentiation and activation receptor(ODAR).
 4. A method of treating an osteopenic disorder in a patient,comprising administering to a patient a pharmaceutical composition,wherein the pharmaceutical composition comprises a pharmaceuticallyacceptable carrier and a therapeutically effective amount of an isolatedhuman antibody that specifically binds osteoprotegerin ligand (OPGL),wherein the antibody comprises: (a) a heavy chain having a heavy chainvariable region comprising an amino acid sequence as set forth in SEQ IDNO: 10, an antigen-binding fragment thereof, or an immunologicallyfunctional immunoglobulin fragment thereof, and a light chain having alight chain variable region comprising an amino acid sequence as setforth in SEQ ID NO: 12, an antigen-binding fragment thereof, or animmunologically functional immunoglobulin fragment thereof; (b) a heavychain having a heavy chain variable region comprising an amino acidsequence as set forth in SEQ ID NO: 18, an antigen-binding fragmentthereof, or an immunologically functional immunoglobulin fragmentthereof, and a light chain having a light chain variable regioncomprising an amino acid sequence as set forth in SEQ ID NO: 20, anantigen-binding fragment thereof, or an immunologically functionalimmunoglobulin fragment thereof; (c) a heavy chain comprising an aminoacid sequence as set forth in SEQ ID NO: 34, an antigen-binding fragmentthereof, or an immunologically functional immunoglobulin fragmentthereof, and a light chain comprising an amino acid sequence as setforth in SEQ ID NO: 36, an antigen-binding fragment thereof, or animmunologically functional immunoglobulin fragment thereof; or (d) aheavy chain comprising an amino acid sequence as set forth in SEQ ID NO:42, an antigen-binding fragment thereof, or an immunologicallyfunctional immunoglobulin fragment thereof, and a light chain comprisingan amino acid sequence as set forth in SEQ ID NO: 44, an antigen-bindingfragment thereof, or an immunologically functional immunoglobulinfragment thereof; wherein the antibody binding inhibits binding of OPGLto an osteoclast differentiation and activation receptor (ODAR).